Processes for production of tumor infiltrating lymphocytes and uses of same in immunotherapy

ABSTRACT

The present invention provides improved and/or shortened methods for expanding TILs and producing therapeutic populations of TILs, including novel methods for expanding TIL populations in a closed system that lead to improved efficacy, improved phenotype, and increased metabolic health of the TILs in a shorter time period, while allowing for reduced microbial contamination as well as decreased costs. Such TILs find use in therapeutic treatment regimens.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/863,634, filed on Jan. 5, 2018, which claims priority to U.S.Provisional Patent Application No. 62/478,506, filed on Mar. 29, 2017,U.S. Provisional Patent Application No. 62/539,410, filed on Jul. 31,2017, U.S. Provisional Patent Application No. 62/548,306, filed on Aug.21, 2017, U.S. Provisional Patent Application No. 62/554,538, filed onSep. 5, 2017, U.S. Provisional Patent Application No. 62/559,374, filedon Sep. 15, 2017, U.S. Provisional Patent Application No. 62/567,121,filed on Oct. 2, 2017, U.S. Provisional Patent Application No.62/577,655, filed on Oct. 26, 2017, U.S. Provisional Patent ApplicationNo. 62/582,874, filed on Nov. 7, 2017, and U.S. Provisional PatentApplication No. 62/596,374, filed on Dec. 8, 2017, which are herebyincorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 5, 2018, isnamed 116983-5017-US ST25.txt and is 14 kilobytes in size.

BACKGROUND OF THE INVENTION

Treatment of bulky, refractory cancers using adoptive transfer of tumorinfiltrating lymphocytes (TILs) represents a powerful approach totherapy for patients with poor prognoses. Gattinoni, et al., Nat. Rev.Immunol. 2006, 6, 383-393. A large number of TILs are required forsuccessful immunotherapy, and a robust and reliable process is neededfor commercialization. This has been a challenge to achieve because oftechnical, logistical, and regulatory issues with cell expansion.IL-2-based TIL expansion followed by a “rapid expansion process” (REP)has become a preferred method for TIL expansion because of its speed andefficiency. Dudley, et al., Science 2002, 298, 850-54; Dudley, et al.,J. Clin. Oncol. 2005, 23, 2346-57; Dudley, et al., J. Clin. Oncol. 2008,26, 5233-39; Riddell, et al., Science 1992, 257, 238-41; Dudley, et al.,J. Immunother. 2003, 26, 332-42. REP can result in a 1,000-foldexpansion of TILs over a 14-day period, although it requires a largeexcess (e.g., 200-fold) of irradiated allogeneic peripheral bloodmononuclear cells (PBMCs, also known as mononuclear cells (MNCs)), oftenfrom multiple donors, as feeder cells, as well as anti-CD3 antibody(OKT3) and high doses of IL-2. Dudley, et al., J. Immunother. 2003, 26,332-42. TILs that have undergone an REP procedure have producedsuccessful adoptive cell therapy following host immunosuppression inpatients with melanoma. Current infusion acceptance parameters rely onreadouts of the composition of TILs (e.g., CD28, CD8, or CD4 positivity)and on fold expansion and viability of the REP product.

Current TIL manufacturing processes are limited by length, cost,sterility concerns, and other factors described herein such that thepotential to commercialize such processes is severely limited, and forthese and other reasons, at the present time no commercial process hasbecome available. There is an urgent need to provide TIL manufacturingprocesses and therapies based on such processes that are appropriate forcommercial scale manufacturing and regulatory approval for use in humanpatients at multiple clinical centers.

BRIEF SUMMARY OF THE INVENTION

The present invention provides improved and/or shortened methods forexpanding TILs and producing therapeutic populations of TILs.

The present invention provides a method for expanding tumor infiltratinglymphocytes (TILs) into a therapeutic population of TILs comprising:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient by processing a tumor sample obtained from the        patient into multiple tumor fragments;    -   (b) adding the tumor fragments into a closed system;    -   (c) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2 to        produce a second population of TILs, wherein the first expansion        is performed in a closed container providing a first        gas-permeable surface area, wherein the first expansion is        performed for about 3-14 days to obtain the second population of        TILs, wherein the second population of TILs is at least 50-fold        greater in number than the first population of TILs, and wherein        the transition from step (b) to step (c) occurs without opening        the system;    -   (d) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        third population of TILs, wherein the second expansion is        performed for about 7-14 days to obtain the third population of        TILs, wherein the third population of TILs is a therapeutic        population of TILs, wherein the second expansion is performed in        a closed container providing a second gas-permeable surface        area, and wherein the transition from step (c) to step (d)        occurs without opening the system;    -   (e) harvesting the therapeutic population of TILs obtained from        step (d), wherein the transition from step (d) to step (e)        occurs without opening the system; and    -   (f) transferring the harvested TIL population from step (e) to        an infusion bag, wherein the transfer from step (e) to (f)        occurs without opening the system.

In some embodiments, the method further comprises the step ofcryopreserving the infusion bag comprising the harvested TIL populationin step (f) using a cryopreservation process.

In some embodiments, the cryopreservation process is performed using a1:1 ratio of harvested TIL population to cryopreservation media.

In some embodiments, the antigen-presenting cells are peripheral bloodmononuclear cells (PBMCs). In some embodiments, the PBMCs are irradiatedand allogeneic. In some embodiments, the PBMCs are added to the cellculture on any of days 9 through 14 in step (d). In some embodiments,the antigen-presenting cells are artificial antigen-presenting cells.

In some embodiments, the harvesting in step (e) is performed using amembrane-based cell processing system.

In some embodiments, the harvesting in step (e) is performed using aLOVO cell processing system.

In some embodiments, the multiple fragments comprise about 4 to about 50fragments, wherein each fragment has a volume of about 27 mm³.

In some embodiments, the multiple fragments comprise about 30 to about60 fragments with a total volume of about 1300 mm³ to about 1500 mm³.

In some embodiments, the multiple fragments comprise about 50 fragmentswith a total volume of about 1350 mm³.

In some embodiments, the multiple fragments comprise about 50 fragmentswith a total mass of about 1 gram to about 1.5 grams.

In some embodiments, the cell culture medium is provided in a containerselected from the group consisting of a G-container and a Xuri cellbag.

In some embodiments, the cell culture medium in step (d) furthercomprises IL-15 and/or IL-21.

In some embodiments, the IL-2 concentration is about 10,000 IU/mL toabout 5,000 IU/mL.

In some embodiments, the IL-15 concentration is about 500 IU/mL to about100 IU/mL.

In some embodiments, the IL-21 concentration is about 20 IU/mL to about0.5 IU/mL.

In some embodiments, the infusion bag in step (f) is aHypoThermosol-containing infusion bag.

In some embodiments, the cryopreservation media comprisesdimethlysulfoxide (DMSO). In some embodiments, the cryopreservationmedia comprises 7% to 10% dimethlysulfoxide (DMSO).

In some embodiments, the first period in step (c) and the second periodin step (e) are each individually performed within a period of 10 days,11 days, or 12 days.

In some embodiments, the first period in step (c) and the second periodin step (e) are each individually performed within a period of 11 days.

In some embodiments, steps (a) through (f) are performed within a periodof about 10 days to about 22 days.

In some embodiments, steps (a) through (f) are performed within a periodof about 20 days to about 22 days.

In some embodiments, steps (a) through (f) are performed within a periodof about 15 days to about 20 days.

In some embodiments, steps (a) through (f) are performed within a periodof about 10 days to about 20 days.

In some embodiments, steps (a) through (f) are performed within a periodof about 10 days to about 15 days.

In some embodiments, steps (a) through (f) are performed in 22 days orless.

In some embodiments, steps (a) through (f) are performed in 20 days orless.

In some embodiments, steps (a) through (f) are performed in 15 days orless.

In some embodiments, steps (a) through (f) are performed in 10 days orless.

In some embodiments, steps (a) through (f) and cryopreservation areperformed in 22 days or less.

In some embodiments, the therapeutic population of TILs harvested instep (e) comprises sufficient TILs for a therapeutically effectivedosage of the TILs.

In some embodiments, the number of TILs sufficient for a therapeuticallyeffective dosage is from about 2.3×1010 to about 13.7×1010.

In some embodiments, steps (b) through (e) are performed in a singlecontainer, wherein performing steps (b) through (e) in a singlecontainer results in an increase in TIL yield per resected tumor ascompared to performing steps (b) through (e) in more than one container.

In some embodiments, the antigen-presenting cells are added to the TILsduring the second period in step (d) without opening the system.

In some embodiments, the third population of TILs in step (d) providesfor increased efficacy, increased interferon-gamma production, increasedpolyclonality, increased average IP-10, and/or increased average MCP-1when adminstered to a subject.

In some embodiments, the third population of TILs in step (d) providesfor at least a five-fold or more interferon-gamma production whenadminstered to a subject.

In some embodiments, the third population of TILs in step (d) is atherapeutic population of TILs which comprises an increasedsubpopulation of effector T cells and/or central memory T cells relativeto the second population of TILs, wherein the effector T cells and/orcentral memory T cells in the therapeutic population of TILs exhibit oneor more characteristics selected from the group consisting of expressingCD27+, expressing CD28+, longer telomeres, increased CD57 expression,and decreased CD56 expression relative to effector T cells, and/orcentral memory T cells obtained from the second population of cells.

In some embodiments, the effector T cells and/or central memory T cellsobtained from the third population of TILs exhibit increased CD57expression and decreased CD56 expression relative to effector T cellsand/or central memory T cells obtained from the second population ofcells.

In some embodiments, the risk of microbial contamination is reduced ascompared to an open system.

In some embodiments, the TILs from step (g) are infused into a patient.

In some embodiments, the multiple fragments comprise about 4 fragments.

The present invention also provides a method for treating a subject withcancer, the method comprising administering expanded tumor infiltratinglymphocytes (TILs) comprising:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a subject by processing a tumor sample obtained from the        patient into multiple tumor fragments;    -   (b) adding the tumor fragments into a closed system;    -   (c) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2 to        produce a second population of TILs, wherein the first expansion        is performed in a closed container providing a first        gas-permeable surface area, wherein the first expansion is        performed for about 3-14 days to obtain the second population of        TILs, wherein the second population of TILs is at least 50-fold        greater in number than the first population of TILs, and wherein        the transition from step (b) to step (c) occurs without opening        the system;    -   (d) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        third population of TILs, wherein the second expansion is        performed for about 7-14 days to obtain the third population of        TILs, wherein the third population of TILs is a therapeutic        population of TILs, wherein the second expansion is performed in        a closed container providing a second gas-permeable surface        area, and wherein the transition from step (c) to step (d)        occurs without opening the system;    -   (e) harvesting the therapeutic population of TILs obtained from        step (d), wherein the transition from step (d) to step (e)        occurs without opening the system; and    -   (f) transferring the harvested TIL population from step (e) to        an infusion bag, wherein the transfer from step (e) to (f)        occurs without opening the system;    -   (g) optionally cryopreserving the infusion bag comprising the        harvested TIL population from step (f) using a cryopreservation        process; and    -   (h) administering a therapeutically effective dosage of the        third population of TILs from the infusion bag in step (g) to        the patient.

In some embodiments, the therapeutic population of TILs harvested instep (e) comprises sufficient TILs for administering a therapeuticallyeffective dosage of the TILs in step (h).

In some embodiments, the number of TILs sufficient for administering atherapeutically effective dosage in step (h) is from about 2.3×1010 toabout 13.7×1010.

In some embodiments, the antigen presenting cells (APCs) are PBMCs.

In some embodiments, the PBMCs are added to the cell culture on any ofdays 9 through 14 in step (d).

In some embodiments, prior to administering a therapeutically effectivedosage of TIL cells in step (h), a non-myeloablative lymphodepletionregimen has been administered to the patient.

In some embodiments, the non-myeloablative lymphodepletion regimencomprises the steps of administration of cyclophosphamide at a dose of60 mg/m2/day for two days followed by administration of fludarabine at adose of 25 mg/m2/day for five days.

In some embodiments, the method further comprises the step of treatingthe patient with a high-dose IL-2 regimen starting on the day afteradministration of the TIL cells to the patient in step (h).

In some embodiments, the high-dose IL-2 regimen comprises 600,000 or720,000 IU/kg administered as a 15-minute bolus intravenous infusionevery eight hours until tolerance.

In some embodiments, the third population of TILs in step (d) is atherapeutic population of TILs which comprises an increasedsubpopulation of effector T cells and/or central memory T cells relativeto the second population of TILs, wherein the effector T cells and/orcentral memory T cells in the therapeutic population of TILs exhibit oneor more characteristics selected from the group consisting of expressingCD27+, expressing CD28+, longer telomeres, increased CD57 expression,and decreased CD56 expression relative to effector T cells, and/orcentral memory T cells obtained from the second population of cells.

In some embodiments, the effector T cells and/or central memory T cellsin the therapeutic population of TILs exhibit increased CD57 expressionand decreased CD56 expression relative to effector T cells and/orcentral memory T cells obtained from the second population of cells.

In some embodiments, the cancer is selected from the group consisting ofmelanoma, ovarian cancer, cervical cancer, non-small-cell lung cancer(NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused byhuman papilloma virus, head and neck cancer (including head and necksquamous cell carcinoma (HNSCC)), renal cancer, and renal cellcarcinoma.

In some embodiments, the cancer is selected from the group consisting ofmelanoma, HNSCC, cervical cancers, and NSCLC.

In some embodiments, the cancer is melanoma.

In some embodiments, the cancer is HNSCC.

In some embodiments, the cancer is a cervical cancer.

In some embodiments, the cancer is NSCLC.

The present invention also provides methods for expanding tumorinfiltrating lymphocytes (TILs) into a therapeutic population of TILscomprising:

-   -   (a) adding processed tumor fragments from a tumor resected from        a patient into a closed system to obtain a first population of        TILs;    -   (b) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2 to        produce a second population of TILs, wherein the first expansion        is performed in a closed container providing a first        gas-permeable surface area, wherein the first expansion is        performed for about 3-14 days to obtain the second population of        TILs, wherein the second population of TILs is at least 50-fold        greater in number than the first population of TILs, and wherein        the transition from step (a) to step (b) occurs without opening        the system;    -   (c) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        third population of TILs, wherein the second expansion is        performed for about 7-14 days to obtain the third population of        TILs, wherein the third population of TILs is a therapeutic        population of TILs, wherein the second expansion is performed in        a closed container providing a second gas-permeable surface        area, and wherein the transition from step (b) to step (c)        occurs without opening the system;    -   (d) harvesting the therapeutic population of TILs obtained from        step (c), wherein the transition from step (c) to step (d)        occurs without opening the system; and    -   (e) transferring the harvested TIL population from step (d) to        an infusion bag, wherein the transfer from step (d) to (e)        occurs without opening the system.

In some embodiments, the therapeutic population of TILs harvested instep (d) comprises sufficient TILs for a therapeutically effectivedosage of the TILs.

In some embodiments, the number of TILs sufficient for a therapeuticallyeffective dosage is from about 2.3×1010 to about 13.7×1010.

In some embodiments, the method further comprises the step ofcryopreserving the infusion bag comprising the harvested TIL populationusing a cryopreservation process.

In some embodiments, the cryopreservation process is performed using a1:1 ratio of harvested TIL population to cryopreservation media.

In some embodiments, the antigen-presenting cells are peripheral bloodmononuclear cells (PBMCs).

In some embodiments, the PBMCs are irradiated and allogeneic.

In some embodiments, the PBMCs are added to the cell culture on any ofdays 9 through 14 in step (c).

In some embodiments, the antigen-presenting cells are artificialantigen-presenting cells.

In some embodiments, the harvesting in step (d) is performed using aLOVO cell processing system.

In some embodiments, the multiple fragments comprise about 4 to about 50fragments, wherein each fragment has a volume of about 27 mm³.

In some embodiments, the multiple fragments comprise about 30 to about60 fragments with a total volume of about 1300 mm³ to about 1500 mm³.

In some embodiments, the multiple fragments comprise about 50 fragmentswith a total volume of about 1350 mm³.

In some embodiments, the multiple fragments comprise about 50 fragmentswith a total mass of about 1 gram to about 1.5 grams.

In some embodiments, the multiple fragments comprise about 4 fragments.

In some embodiments, the second cell culture medium is provided in acontainer selected from the group consisting of a G-container and a Xuricellbag.

In some embodiments, the infusion bag in step (e) is aHypoThermosol-containing infusion bag.

In some embodiments, the first period in step (b) and the second periodin step (c) are each individually performed within a period of 10 days,11 days, or 12 days.

In some embodiments, the first period in step (b) and the second periodin step (c) are each individually performed within a period of 11 days.

In some embodiments, steps (a) through (e) are performed within a periodof about 10 days to about 22 days.

In some embodiments, steps (a) through (e) are performed within a periodof about 10 days to about 20 days.

In some embodiments, steps (a) through (e) are performed within a periodof about 10 days to about 15 days.

In some embodiments, steps (a) through (e) are performed in 22 days orless.

In some embodiments, steps (a) through (e) and cryopreservation areperformed in 22 days or less.

In some embodiments, steps (b) through (e) are performed in a singlecontainer, wherein performing steps (b) through (e) in a singlecontainer results in an increase in TIL yield per resected tumor ascompared to performing steps (b) through (e) in more than one container.

In some embodiments, the antigen-presenting cells are added to the TILsduring the second period in step (c) without opening the system.

In some embodiments, the third population of TILs in step (d) is atherapeutic population of TILs which comprises an increasedsubpopulation of effector T cells and/or central memory T cells relativeto the second population of TILs, wherein the effector T cells and/orcentral memory T cells obtained in the therapeutic population of TILsexhibit one or more characteristics selected from the group consistingof expressing CD27+, expressing CD28+, longer telomeres, increased CD57expression, and decreased CD56 expression relative to effector T cells,and/or central memory T cells obtained from the second population ofcells.

In some embodiments, the effector T cells and/or central memory T cellsobtained in the therapeutic population of TILs exhibit increased CD57expression and decreased CD56 expression relative to effector T cells,and/or central memory T cells obtained from the second population ofcells.

In some embodiments, the risk of microbial contamination is reduced ascompared to an open system.

In some embodiments, the TILs from step (e) are infused into a patient.

In some embodiments, the closed container comprises a single bioreactor.

In some embodiments, the closed container comprises a G-REX-10.

In some embodiments, the closed container comprises a G-REX-100.

In some embodiments, at step (d) the antigen presenting cells (APCs) areadded to the cell culture of the second population of TILs at a APC:TILratio of 25:1 to 100:1.

In some embodiments, the cell culture has a ratio of 2.5×10⁹ APCs to100×10⁶ TILs.

In some embodiments, at step (c) the antigen presenting cells (APCs) areadded to the cell culture of the second population of TILs at a APC:TILratio of 25:1 to 100:1.

In some embodiments, the cell culture has ratio of 2.5×10⁹ APCs to100×10⁶ TILs.

The present invention also provides a population of expanded TILs foruse in the treatment of a subject with cancer, wherein the population ofexpanded TILs is a third population of TILs obtainable by a methodcomprising:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a subject by processing a tumor sample obtained from the        patient into multiple tumor fragments;    -   (b) adding the tumor fragments into a closed system;    -   (c) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2 to        produce a second population of TILs, wherein the first expansion        is performed in a closed container providing a first        gas-permeable surface area, wherein the first expansion is        performed for about 3-14 days to obtain the second population of        TILs, wherein the second population of TILs is at least 50-fold        greater in number than the first population of TILs, and wherein        the transition from step (b) to step (c) occurs without opening        the system;    -   (d) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        third population of TILs, wherein the second expansion is        performed for about 7-14 days to obtain the third population of        TILs, wherein the third population of TILs is a therapeutic        population of TILs, wherein the second expansion is performed in        a closed container providing a second gas-permeable surface        area, and wherein the transition from step (c) to step (d)        occurs without opening the system;    -   (e) harvesting the therapeutic population of TILs obtained from        step (d), wherein the transition from step (d) to step (e)        occurs without opening the system; and    -   (f) transferring the harvested TIL population from step (e) to        an infusion bag, wherein the transfer from step (e) to (f)        occurs without opening the system; and    -   (g) optionally cryopreserving the infusion bag comprising the        harvested TIL population from step (f) using a cryopreservation        process.

In some embodiments, the population of TILs is for use to treat asubject with cancer according the methods described above and herein,wherein the method further comprises one or more of the features recitedabove and herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Shows a diagram of an embodiment of process 2A, a 22-day processfor TIL manufacturing.

FIG. 2: Shows a comparison between the 1C process and an embodiment ofthe 2A process for TIL manufacturing.

FIG. 3: Shows the 1C process timeline.

FIG. 4: Shows the process of an embodiment of TIL therapy using process2A for TIL manufacturing, including administration and co-therapy steps,for higher cell counts.

FIG. 5: Shows the process of an embodiment of TIL therapy using process2A for TIL manufacturing, including administration and co-therapy steps,for lower cell counts.

FIG. 6: Shows a detailed schematic for an embodiment of the 2A process.

FIG. 7: Shows characterization of TILs prepared using an embodiment ofthe 2A process by comparing interferon-gamma (IFN-γ) expression betweenfresh TILs and thawed TILs.

FIG. 8: Shows characterization of TILs prepared using an embodiment ofthe 2A process by examining CD3 expression in fresh TILs versus thawedTILs.

FIG. 9: Shows characterization of TILs prepared using an embodiment ofthe 2A process by examining recovery in fresh TILs versus thawed TILs.

FIG. 10: Shows characterization of TILs prepared using an embodiment ofthe 2A process by examining viability of fresh TILs versus thawed TILs.

FIG. 11A, FIG. 11B, and FIG. 11C: Depict the major steps of anembodiment of process 2A including the cryopreservation steps.

FIG. 12: Depicts cell counts obtained from the 1C process and anembodiment of the 2A process.

FIG. 13: Depicts percent cell viability obtained from the 1C process andan embodiment of the 2A process.

FIG. 14: Depicts percentages of CD45 and CD3 cells (i.e., T cells)measured by flow cytometry for TILs obtained for the 1C process and anembodiment of the 2A process.

FIG. 15: Depicts IFN-γ release obtained for the 1C process andembodiments of the 2A process, as measured by an assay different thanthat used to generate the data in FIGS. 80 and 98.

FIG. 16: Depicts IFN-γ release obtained for the 1C process andembodiments of the 2A process, as measured by an assay different thanthat used to generate the data in FIGS. 80 and 98.

FIG. 17: Depicts percentages of TCR a/b and NK cells obtained from the1C process and an embodiment of the 2A process.

FIG. 18: Depicts percentages of CD8⁺ and CD4⁺ cells measured by flowcytometry for TILs obtained by the 1C process and an embodiment of the2A process, as well as the ratio between each subset.

FIG. 19: Depicts percentages of memory subsets measured by flowcytometry for TILs obtained from the 1C process and an embodiment of the2A process.

FIG. 20: Depicts percentages of PD-1, LAG-3, and TIM-3 expression byflow cytometry for TILs obtained from the 1C process and an embodimentof the 2A process.

FIG. 21: Depicts percentages of 4-1BB, CD69, and KLRG1 expression byflow cytometry for TILs obtained from the 1C process and an embodimentof the 2A process.

FIG. 22: Depicts percentages of TIGIT expression by flow cytometry forTILs obtained from the 1C process and an embodiment of the 2A process.

FIG. 23: Depicts percentages of CD27 and CD28 expression by flowcytometry for TILs obtained from the 1C process and an embodiment of the2A process.

FIG. 24: Depicts the results of flow-FISH telomere length analysis.

FIG. 25: Depicts the results of flow-FISH telomere length analysis(after removal of an outlier data point).

FIG. 26: Depicts the clinical trial design including cohorts treatedwith process 1C and an embodiment of process 2A.

FIG. 27: Exemplary Process 2A chart providing an overview of Steps Athrough F.

FIG. 28A, FIG. 28B and FIG. 28C: Process Flow Chart of Process 2A.

FIG. 29: Process Flow Chart on Process 2A Data Collection Plan

FIG. 30: Viability of fresh vs. thawed TIL

FIG. 31: Expansion of fresh and thawed TIL in re-REP culture

FIG. 32: Normal laboratory values of blood metabolites.

FIG. 33A and FIG. 33B: Metabolite analysis of process 2A pre-REP TIL.

FIG. 34: Quantification of IL-2 in process 2A pre-REP TIL cell culture.

FIG. 35: Release of cytotoxic cytokines IFN-γ upon anti-CD3, anti-CD28and anti-4-1BB stimulation of TIL.

FIG. 36: Release of Granzyme B following anti-CD3, anti-CD28, andanti-4-1BB stimulation of TIL.

FIG. 37A and FIG. 37B: TCR αβ+ TIL. Most human CD3+ T-cells express thereceptors formed by α and β chains that recognize antigens in an MHCrestricted manner. A) Except in M1061, fresh and thawed TIL product had80% or more TCR αβ+ expressing TIL. Both fresh and thaw TIL hadcomparable expression of TCR αβ (p-value—0.9582). Even though a decreasein the TCR αβ+ expressing TIL after the Re-REP was observed, thisdecrease was not significant within the Re-REP TIL (p=0.24). B) Therewas a 9.2% and 15.7% decrease in the fresh and thaw RE-REP TILexpressing TCR αβ in comparison to fresh and thaw TIL respectively.

FIG. 38A and FIG. 38B: TCRαβ-CD56+. Tumor infiltrating Natural Killer(NK) and NKT-cells also have the ability to lyse cells lacking MHCexpression as well as CD1-presented lipid antigen and to provideimmunoregulatory cytokines. However, an intense NK cell infiltration isassociated with advanced disease and could facilitate cancerdevelopment. Figure A shows that in all instances, except in M1063,there was a modest, though not significant, decrease in NK population inthawed TIL compared to fresh TIL, (p=0.27). No significant differencewas observed between the re-REP TIL population (p=0.88). Fresh TIL,fresh re-REP TIL, and thawed re-REP TIL demonstrate similar expressionof CD56 as shown in Figure B. Thawed TIL product had less (1.9±1.3)NK-expressing cells than fresh TIL (3.0±2.2) possibly as a result of thecryo-freezing procedure.

FIG. 39A and FIG. 39B: CD4+ cells. No substantial difference in the CD4population was observed in individual conditions. Figure A representsthe average CD4 population in each condition. The table in Figure Bshows the SD and SEM values. There is a slight decrease in the CD4population in the fresh re-REP population which is mostly due to adecrease in CD4 in the fresh re-REP population in EP11001T.

FIG. 40A and FIG. 40B: CD8+ cells. A) In all, except EP11001T, bothfresh and thawed TIL showed comparable CD8+ populations (p=0.10, nosignificant difference). In most experiments, there was a slightdecrease in the CD8+ expressing TIL in the fresh re-REP TIL product(exceptions were M1061T and M1065T). There was approximately a 10-30%decrease in the CD8+ population in the thawed re-REP TIL. Comparison ofthe re-REP TIL from both fresh and thawed TIL showed a significantdifference (p=0.03, Student's t-test). Figure B shows the mean values ofthe CD8+ expressing TIL in all conditions. Both fresh and thawed TILshow similar results. However, there was a 10.8% decrease in the CD8+population in the thawed re-REP TIL product in comparison to the freshre-REP TIL.

FIG. 41A and FIG. 41B: CD4+CD154+ cells. CD154, also known as CD40L is amarker for activated T-cells. Figure A: No substantial difference in theCD4+CD154+ population was observed in the different conditions, however,a decrease of 34.1% was observed in the EP11001T fresh re-REP CD4+ TILs.CD154 expression were not measured in M1061T and M1062T as theseexperiments were carried out before the extended phenotype panel was inplace. Figure B: A slight decrease in thawed TIL condition could beattributed to CD154 not measured in M1061T and M1062T. All conditionsshow very comparable CD154 expression in the CD4 population suggestingactivated CD4+ T cells.

FIG. 42A and FIG. 42B: CD8+CD154+ cells. Activation marker CD154expressed on CD8+ TIL was also analyzed. A) Overall, the CD154expression was lower in the CD8+ population in the fresh and thawed TILproduct. This is not surprising as CD154 is expressed mainly in theactivated CD4+ T cells. In cases where the CD154 expression was measuredin both fresh and thawed TIL product, either a no difference or anincrease in the CD154 expression was observed in the thawed TILproducts. Student's t-test showed the there was no significantdifference between the two conditions. An increase in the CD154expression in the thawed re-REP in comparison to the fresh re-REP wasshown in all experiments (p=0.02). B) An increase in CD154 expressionwas observed in both the thawed TIL and thawed re-REP TIL products incomparison to their counterparts. Thawed re-REP TIL showed a 29.1%increase in CD154 expression compared to the fresh re-REP TIL.

FIG. 43A and FIG. 43B: CD4+CD69+ cells. CD69 is the early activationmarker in T cell following stimulation or activation. A) In all TILexcept in EP11001T, both fresh and thawed re-REP showed a modestincrease in CD69 expression, possibly due to the re-REP length (7 daysrather than 11 days). No difference was observed between fresh andthawed TIL (p=0.89). A difference between fresh and thawed re-REP wasalso not observed (p=0.82). B) A minor increase in CD69 expression isobserved in the re-REP TIL products. (Note: No CD69 staining wasperformed for either M1061T and M1062T thawed TIL product. CD69expression of M1061T fresh TIL product was 33.9%).

FIG. 44A and FIG. 44B: CD8+CD69+ cells. As observed for the CD4+population, Figure A shows an increase in the CD69 expression in theCD8+re-REP TIL. CD69 expression showed no significant difference betweenthe fresh and thawed TIL (p=0.68) or the fresh and thawed re-REP TIL(p=0.76). Figure B supports the observation that there is a modestincrease in the CD69 expression in the re-REP TIL product.

FIG. 45A and FIG. 45B: CD4+CD137+ cells. CD137 (4-11313) is a T-cellcostimulatory receptor induced upon TCR activation. It is activated onCD4+ and CD8+ T cells. A) CD137 expression showed a profound increase inthe re-REP TIL population following 7 days of stimulation. However, nodifference between the fresh and thawed TIL or fresh and thawed re-REPTIL were observed (p<0.05 in both cases Figure B supports thisobservation). Also, the thawed TIL showed a modest decrease in CD137expression. The increase in CD137 expression in re-REP TIL could beattributed to the second round of stimulation of the 7-day re-REP.

FIG. 46A and FIG. 46B: CD8+CD137+ cells. A) CD8+ population showed anoverall increase in the re-REP product. B) Fresh re-REP product had a33.4% increase in CD8+CD137+ expression in comparison to fresh TILproduct. Thawed re-REP product also showed a 33.15% increase in CD137expression in the CD8+ population compared to thawed TIL. No significantdifferences were observed between fresh and thawed re-REP TIL. A similarobservation can be seen comparing the fresh TIL to the thawed TILproduct. This increase in CD137 expression could be due to the secondround of activation of the re-REP. (Note that only 6 TIL were used forthe analysis as CD137 expression were not measured for 3 of theexperiments.)

FIG. 47A and FIG. 47B: CD4+CM cells. Central Memory (CM) population isdefined by CD45RA− (negative) and CCR7+(positive) expression. A) Anincrease in the CM population in the re-REP conditions were observed.M1063T and M1064T showed a decrease in the CM expression in the CD4+population obtained from thawed TIL in comparison to fresh TIL product.Neither fresh and thawed TIL product (p=0.1658) nor fresh re-REP andthaw re-REP TIL (p=0.5535) showed a significant difference in CMpopulation. B) A 14.4% and 15.4% increase in the CM population wasobserved in the fresh and thawed re-REP TIL in comparison to fresh andthawed TIL respectively.

FIG. 48A and FIG. 48B: CD8+CM cells. A) In the CD8+ population, adramatic increase in CM expression in the fresh TIL product was seen, anobservation not present in the TIL product. This increase did not affectthe significance (p=0.3086), suggesting no difference between the freshand thawed TIL. A similar trend was seen in the re-REP TIL products aswell. FIG. 48B) An overall increase in CM population in the fresh TILwas observed in comparison to the thawed TIL. The numbers show thatfresh TIL and re-REP TIL had only a difference of ˜2%; the fresh TILshowed a very high standard deviation which could be attributed toM1064T; excluding the CM expression in M1064T resulted in very similarCM expression between the fresh and thawed TIL product (not shown).

FIG. 49A and FIG. 49B: CD4+EM cells. Effector memory (EM) population isdefined by the lack of CCR7 and CD45RA expression. A) As expected theCD4+ population from fresh and thawed TIL had a high level of effectormemory phenotype. A drastic decrease in the effector memory expressionwas found in the M1056T re-REP TIL population. Also, 5 other experimentsshowed a decrease in the effector memory phenotype in both fresh andthawed re-REP TIL. B) Both fresh and thawed TIL showed similarexpression of effector memory phenotype. Comparison of fresh and freshRe-REP TIL showed a decrease by 16% in the latter. A similar decreasewas observed in the thawed Re-REP TIL (9%) when compared to the thawedTIL.

FIG. 50A and FIG. 50B: CD8+EM cells. A) A similar pattern of increasedeffector memory in the fresh TIL was also seen in the CD8+ population.An exception was noted in the M1064T in which fresh TIL only had a 20%effector memory profile; this is due to the 73% of these TIL having a CMphenotype as described in A and B. All the samples showing a decrease inthe effector memory population in their CD4+ TIL from the re-REP productfollowed the same trend in their CD8+ TIL. B) Unlike the CD4+ TILpopulation, CD8+ TIL showed a similar effector memory phenotype infresh, thawed and re-REP products. (Note the high standard deviation inthe fresh and thawed TIL, which are due to the low effector memorypopulation in M1064T fresh and to no expression in M1061T thawed TILsamples.)

FIG. 51A and FIG. 51B: CD4+CD28+ cells. CD28 expression correlates withyoung TIL decreasing with age. A) Even though an increase in the CMpopulation was observed in the re-REP TIL, a decrease in the CD28expression was seen as a trend suggesting that CM-status alone could notdetermine the fate of TIL. A decrease in CD28 expression was observed inthe -re-REP product, except for M1061T CD4+ TIL. B) A decrease of 8.89%in the fresh and 5.71% in the thawed TIL was seen compared to fresh andthawed TIL product, respectively.

FIG. 52A and FIG. 52B: CD8+CD28+ cells. A) CD28 expression in the CD8+TIL population was higher in the fresh and thawed TIL than re-REPproduct. In most cases, thawed re-REP TIL showed a drastic decrease whencompared to thawed TIL and fresh re-REP TIL. However, Student's t-testshowed no significant difference between fresh and thawed TIL (p=0.3668)and also between the fresh and thawed re-REP products (p−=0.7940). B) Asseen in the CD4+ TIL population, there was a decrease in CD8+CD28+populations in the fresh re-REP (21.5%) and thawed re-REP (18.2%) whencompared to their non-restimulated counterparts.

FIG. 53A and FIG. 53B: CD4+PD-1+ cells. PD-1 expression in TIL iscorrelated with antigen reactive and exhausted T cells. I Thus it is notsurprising that an exhausted phenotype is observed in TIL which haveundergone a REP for 11 days. A) This exhausted phenotype was eithermaintained or increased (specifically, EP11001T and M1056T) in thethawed TIL product. No significant difference between fresh and thawedTIL product was seen (p=0.9809). A similar trend was shown in the freshcompared to thawed re-REP TIL (p=0.0912). B) Fresh re-REP showed amodest decrease in PD-1 expression in the CD4+ TIL population. All theother conditions maintained a comparable PD-1 expression pattern. Adecrease or no change in PD-1 expression was observed in fresh re-REPproduct compared to all other conditions. An increase in the PD-1expression was seen in M1062T, M1063T (CD4+) and EP (CD8+) in the thawedre-REP product. All other thawed re-REP product showed comparableresults to the thawed product.

FIG. 54A and FIG. 54B: CD8+PD-1+ cells. A) CD8+ population from thefresh TIL product showed a more exhausted phenotype associated withincreased PD-1 expression. An exception was observed in EP11001T whereCD8+ thawed TIL product had a modest increase in the PD-1 expressioncompared to fresh TIL product. There was a small, though non-significantdifference in the PD-1 expression in the fresh TIL compared to thawedTIL (p=0.3144). B) Fresh TIL product showed a slight increase, butnon-significant PD-1 expression compared to thawed TIL (6.74%, or1.2-fold higher than thawed TIL) suggesting that the thawed TIL productwas comparable based on the phenotype pattern.

FIG. 55A and FIG. 55B: CD4+LAG3+ cells. Exhausted T cells express highlevels of inhibitory receptor LAG3 along with PD-1. A) The CD4+ thawedTIL showed slightly higher, but non-significant, levels of LAG3expression in comparison to the fresh TIL (p=0.52). An exception wasobserved in M1063T. In experiments where LAG3 expression in the CD4+fresh and fresh re-REP TIL were measured, a decrease in LAG3+ expressionwas observed in the fresh re-REP samples compared to fresh TIL. B)Overall, there is a modest decrease in the LAG3 expression in freshre-REP TIL product. Please note that for Figure B to maintainconsistent, M1061T, M1062T and M1064T were excluded as LAG3 expressionwere not measured in the fresh product.

FIG. 56A and FIG. 56B: CD8+LAG3+ cells. A) CD8+LAG3+ expressing TILshowed a modest decrease in the experiments, with the exception ofM1063T in which a marked decrease in LAG3 expression was seen in thefresh re-REP TIL. Overall, thawed re-REP TIL showed a 1.5-fold,significant increase compared to fresh re-REP TIL for LAG3 expression(p=0.0154). However, no significant difference was observed betweenfresh TIL and thawed TIL products (p=0.0884). B) An approximate 30%decrease in LAG3 expression in the CD8+ TIL from fresh re-REP wasobserved in comparison to thawed TIL product. Both fresh and thawed TILwere comparable to thawed TIL showing a modest increase. (In thisfigure, M1061T, M1062T and M1064T were omitted as LAG3 expression wasnot measured in the either the fresh or fresh re-REP TIL samples.)

FIG. 57A and FIG. 57B: CD4+TIM-3+ cells. A) As observed previously inthe case of PD-1 and LAG3, a decrease in TIM-3 expression was seen inthe fresh reREP TIL compared to thawed re-REP TIL. Regardless, nosignificant difference existed between fresh and thawed reREP TIL(p=0.2007). B) No major changes in TIM-3 expression was observed amongfresh, thaw and thawed reREP TIL products. A modest decrease of 9.2% inTIM-3 expression was observed in the fresh reREP TIL in comparison tothawed reREP product.

FIG. 58A and FIG. 58B: CD8+TIM-3+ cells. A) A similar trend in TIM-3expression that was seen in the CD4+ population was also seen in theCD8+ TIL. Fresh re-REP TIL had the least exhausted phenotype with lowTIM-3 expression, showing a significant difference in comparison tothawed re-REP TIL (p=0.0147). Comparison of PD-1, LAG3 and TIM-3suggests that fresh re-REP TIL had a less exhaustive phenotype withincreased CM phenotype. B) In comparison to thawed re-REP TIL product,fresh re-REP TIL showed a significant 22% decrease in TIM-3 expression.Both fresh and thawed TIL show similar TIM-3 expression patterns.

FIG. 59: Cytotoxic potential of TIL against P815 target cell line.

FIG. 60A, FIG. 60B, FIG. 60C, FIG. 60D, FIG. 60E, and FIG. 60F:Metabolic respiration profile of fresh TIL, fresh re-REP TIL, and thawedre-REP TIL. Basal OCR (A), Overt SRC (B), SRC2DG (C), Covert SRC (D),Basal ECAR (E), and Glycolytic Reserve (F).

FIG. 61A and FIG. 61B: Flow-FISH technology was used to measure averagelength of Telomere repeat in 9 post-REP Process 2A thawed TIL products.A) Data represents the telomere length measured by qPCR comparing TIL to1301 cells B) Data shows the telomere length measured by Flow Fish Assayof TIL compared to 1301 cells. Data used for graphs are provided in atable format (Tables 25) in the appendix section 10. Overall, there wasa rough similarity in the patterns of the results of the two telomerelength assays, but experiments will continue to determine which methodmore accurately reflects the actual telomere length of the TIL. Thistechnique could be applied to future clinical samples to determine arelationship between telomere length and patient response to TILtherapy.

FIG. 62A and FIG. 62B: Selection of Serum Free Media purveyor (Serumreplacement). Each fragment were cultured in single well of G-Rex 24well plate in quatraplicates. On Day 11, REP were initiated using 4⁵ TILwith 10⁶ Feeders to mimic 2A process. A) Bar graph showing averageviable cell count recorded on Day 11 (preREP) for each conditions. B)Bar graph displaying average viable cell count recorded on Day 22(postREP). P value were calculated using student ‘t’ test. * P<0.05, **P<0.01, *** P<0.001 respectively.

FIG. 63A and FIG. 63B: Selection of Serum Free Media purveyor (PlateletLysate serum). Each fragment were cultured in single well of G-Rex 24well plate in triplicates. On Day 11, REP were initiated using 4e5 TILwith 10e6 Feeders to mimic 2A process. A) Bar graph showing averageviable cell count recorded on Day 11 (preREP) for each conditions. B)Bar graph displaying average viable cell count recorded on Day 22(postREP). P value were calculated using student ‘t’ test. * P<0.05, **P<0.01, *** P<0.001 respectively. ‘#’ Not enough tumor fragments.

FIG. 64A and FIG. 64B: Compare the efficacy of CTS Optimizer withstandard condition using mini scale 2A process (G-Rex 5M). Twofragments/G-Rex 5M were cultured in triplicates, REP were initiatedusing 2⁶ TIL with 50⁶ Feeders to mimic 2A process. Bar presented abovewere average viable cell count obtained on Day 11 (A) or Day 22 (B).

FIG. 65A, FIG. 65B, and FIG. 65C: Summary of pre and post TIL expansionextrapolated comparing standard condition and CTS Optimizer. A) PreREP.B) PostREP. C) Summary of TIL expansion extrapolated to full scale run(Standard vs CTS Optimizer+SR).

FIG. 66: CD8+ was gated on live cells. 7 of the 9 tumors show anincrease in absolute CD8+ populations with the CTS+SR condition.

FIG. 67: Interferon-gamma Comparability. Interferon-gamma ELISA(Quantikine). Production of IFN-γ was measured using Quantikine ELISAkit by R&D systems. CTS+SR produced comparable amounts of IFN-γ whencompared to our standard condition.

FIG. 68: Scheme of on exemplary embodiment of the Rapid ExpansionProtocol (REP). Upon arrival the tumor is fragmented, placed into G-Rexflasks with IL-2 for TIL expansion (pre-REP expansion), for 11 days. Forthe triple cocktail studies, IL-2/IL-15/IL-21 is added at the initiationof the pre-REP. For the Rapid Expansion Protocol (REP), TIL are culturedwith feeders and OKT3 for REP expansion for an additional 11 days.

FIG. 69A and FIG. 69B: TIL derived from melanoma (n=4), and lung (n=7)were assessed phenotypically for CD4+ and CD8+ cells using flowcytometry post pre-REP. *P-values represent the difference between theIL-2 and IL-12/IL-15/IL-21 in the CD8+ cells using student's unpaired ttest.

FIG. 70A and FIG. 70B: TIL derived from melanoma (n=4), and lung (n=7)were assessed phenotypically for CD27+ and CD28+ in the CD4+ and CD8+cells using flow cytometry post pre-REP.

FIG. 71A, FIG. 71B, and FIG. 71C: TIL were assessed phenotypically foreffector/memory subsets (CD45RA and CCR7) in the CD8+ cells andCD4+(data not shown) in melanoma (n=4) (A) and lung (n=8) (B). CXCR3expression was assessed in melanoma and lung. All phenotypic expressionwas assessed using flow cytometry post pre-REP. TCM=central memory,TSCM=stem cell memory, TEMRA (effector T cells), TEM=effector memory.

FIG. 72A, FIG. 72B, and FIG. 72C: TIL derived from (A) melanoma (n=4)and (B) lung (n=5) were assessed for CD107a+ expression in response toPMA stimulation for 4 hours in the CD4+ and CD8+ cells, by flowcytometry. (C) pre-REP TIL (n=5) were stimulated for 24 hours withsoluble OKT3 (30 ng/ml) and the supernatants assessed for IFNγ by ELISA.

FIG. 73A and FIG. 73B: The TCRvβ repertoire (24 specificities) wereassessed in the TIL derived from melanoma (A) and lung (B) using theBeckman Coulter kit for flow cytometry.

FIG. 74: Cryopreserved TIL exemplary manufacturing process (˜22 days).

FIG. 75A and FIG. 75B: On Day 22 the volume reduced cell product ispooled and sampled to determine culture performance prior to wash andformulation. Samples are analyzed on the NC-200 automated cell counteras previously described. Total viable cell density is determined by thegrand mean of duplicate counts from 4 independent samples. TheGeneration 2 (Gen 2) process yields a TIL product of similar dose toGeneration 1 (Gen 1; the Gen 1 mean=4.10×10¹⁰±2.92×10¹⁰, Gen 2mean=3.12×10¹⁰±2.19×10¹⁰. B) Fold expansion is calculated for the REPphase as the dividend of the final viable cell density over the initialviable TIL seeding density. Gen 2 TIL products have a lower foldexpansion relative to Gen 1 (Gen 1 mean=1.40×10³±9.86×10², Gen 2mean=5.11×10²±2.95×10²).

FIG. 76: Fresh formulated drug products were assayed for identity byflow cytometry for release. Gen 1 and Gen 2 processes produce highlypurity T-cell cultures as defined by CD45, CD3 double positive phenotype(Gen 1 #±SD, Gen 2 #±SD). P-value was calculated using Mann-Whitney ‘t’test.

FIG. 77A and FIG. 77B: Cryo preserved satellite vials of formulated drugproduct were thawed and assayed for extended phenotype by flow cytometryas previously described. Gen 1 and Gen 2 products express similar ratiosof CD8 to CD4 T-cell subtypes. P-value was calculated using Mann-WhitneyT test.

FIG. 78A and FIG. 78B: Cryo preserved satellite vials of formulated drugproduct were thawed and assayed for extended phenotype by flow cytometryas previously described. Gen 1 and Gen 2 products express similar levelsof costimulatory molecules CD27 and CD28 on T-cell subsets. P value wascalculated using Mann-Whitney ‘t’ test. Costimulatory molecules such asCD27 and CD28 are required to supply secondary and tertiary signalingnecessary for effector cell proliferation upon T-cell receptorengagement.

FIG. 79: Flow-FISH technology was used to measure the average length ofthe Telomere repeat as previously described. The above RTL valueindicates that the average telomere fluorescence per chromosome/genomein Gen 1 (an embodiment of process 1C) is # %±SD %, and Gen 2 is #%±SD %of the telomere fluorescence per chromosome/genome in the control cellsline (1301 Leukemia cell line). Data indicate that Gen 2 products onaverage have at least comparable telomere lengths to Gen 1 products.Telomere length is a surrogate measure of the length of ex vivo cellculture.

FIG. 80: Gen 2 (an embodiment of the process 2A) drug products exhibitand increased capability of producing IFN-γ relative to Gen 1 drugproducts. The ability of the drug product to be reactivated and secretecytokine is a surrogate measure of in-vivo function upon TCR binding tocognate antigen in the context of HLA.

FIG. 81A and FIG. 81B: T-cell receptor diversity: RNA from 10×10⁶ TILfrom Gen 1 (an embodiment of the process 1C) and Gen 2 (an embodiment ofthe process 2A) drug products were assayed to determine the total numberand frequency of unique CDR3 sequences present in each product. A) Thetotal number of unique CDR3 sequences present in each product (Gen 1n=#, mean±SD, Gen 2 n=#, mean±SD). B) Unique CDR3 sequences were indexedrelative to frequency in each product to yield a score representative ofthe relative diversity of T-cell receptors in the product. TIL productsfrom both processes are composed of polyclonal populations of T-cellswith different antigen specificities and avidities. The breadth of thetotal T-cell repertoire may be indicative of the number of actionableepitopes on tumor cells.

FIG. 82: Shows a diagram of an embodiment of process 2A, a 22-dayprocess for TIL manufacturing.

FIG. 83: Comparison table of Steps A through F from exemplaryembodiments of process 1C and process 2A.

FIG. 84: Detailed comparison of an embodiment of process 1C and anembodiment of process 2A.

FIG. 85: Detailed scheme of an embodiment of a TIL therapy process.

FIGS. 86A, FIG. 86B, and FIG. 86C: Phenotypic characterization of TILproducts using 10-color flow cytometry assay. (A) Percentage of T-celland non-T-cell subsets is defined by CD45⁺CD3⁺ andCD45-(non-lymphocyte)/CD45+CD3⁻ (non-T-cell lymphocyte), respectively.Overall, >99% of the TIL products tested consisted of T-cell(CD45⁺CD3⁺). Shown is an average of TIL products (n=10). (B) Percentageof two T-cell subsets including CD45⁺CD3⁺CD8⁺ (blue open circle) andCD45⁺CD3⁺CD4⁺ (pink open circle). No statistical difference inpercentage of both subsets is observed using student's unpaired T test(P=0.68). (C) Non-T-cell population was characterized for four differentsubsets including: 1) Non-lymphocyte (CD45⁻), 2) NK cell(CD45⁺CD3⁻CD16⁺/56⁺), 3) B-cell (CD45⁺CD19⁺), and 4) Non-NK/B-cell(CD45⁺CD3⁻CD16⁻CD56⁻CD19⁻).

FIG. 87A and FIG. 87B: Characterization of T-cell subsets inCD45+CD3+CD4+ and CD45+CD3+CD8+ cell populations. Naïve, central memory(TCM), effector memory (TEF), and effector memory RA+(EMRA) T-cellsubsets were defined using CD45RA and CCR7. Figures show representativeT-cell subsets from 10 final TIL products in both CD4+(A), and CD8+(B)cell populations. Effector memory T-cell subset (blue open circle) is amajor population (>93%) in both CD4+ and CD8+ subsets of TIL finalproduct. Less than 7% of the TIL products cells is central memory subset(pink open circle). EMRA (gray open circle) and naïve (black opencircle) subsets are barely detected in TIL product (<0.02%). p valuesrepresent the difference between EM and CM using student's unpaired Ttest

FIG. 88A and FIG. 88B: Detection of MCSP and EpCAM expression inmelanoma tumor cells. Melanoma tumor cell lines (WM35, 526, and 888),patient-derived melanoma cell lines (1028, 1032, and 1041), and acolorectal adenoma carcinoma cell line (HT29 as a negative control) werecharacterized by staining for MCSP (melanoma-associated chondroitinsulfate proteoglycan) and EpCAM (epithelial cell adhesion molecule)markers. (A) Average of 90% of melanoma tumor cells express MCSP. (B)EpCAM expression was not detected in melanoma tumor cell lines ascompared positive control HT29, an EpCAM+ tumor cell line.

FIG. 89A and FIG. 89B: Detection of spiked controls for thedetermination of tumor detection accuracy. The assay was performed byspiking known amounts of tumor cells into PBMC suspensions (n=10).MCSP+526 melanoma tumor cells were diluted at ratios of 1:10, 1:100, and1:1,000, then mixed with PBMC and stained with anti-MCSP and anti-CD45antibodies and live/dead dye and analyzed by flow cytometry. (A)Approximately 3000, 300, and 30 cells were detected in the dilution of1:10, 1:100, and 1:1000, respectively. (B) An average (AV) and standarddeviation (SD) of cells acquired in each condition was used to definethe upper and lower reference limits.

FIG. 90A and FIG. 90B: Repeatability study of upper and lower limits inspiked controls. Three independent experiments were performed intriplicate to determine the repeatability of spiking assay. (A) Thenumber of MCSP⁺ detected tumor cells were consistently within the rangeof upper and lower reference limits. (B) Linear regression plotdemonstrates the correlation between MCSP⁺ cells and spiking dilutions(R²=0.99) with the black solid line showing the best fit. The green andgray broken lines represent the 95% prediction limits in standard curveand samples (Exp #1 to 3), respectively.

FIG. 91A and FIG. 91B: Detection of residual melanoma tumor in TILproducts. TIL products were assessed for residual tumor contaminationusing the developed assay (n=15). (A and B) The median number andpercentage of detectable MCSP+ events was 2 and 0.0002%, respectively.

FIG. 92: Potency assessment of TIL products following T-cell activation.IFNγ secretion after re-stimulation with anti-CD3/CD28/CD137 in TILproducts assessed by ELISA in duplicate (n=5). IFNγ secretion by the TILproducts was significantly greater than unstimulated controls usingWilcoxon signed rank test (P=0.02), and consistently >1000 pg/ml. IFNγsecretion>200 pg/ml is considered to be potent. p value<0.05 isconsidered statistically significant.

FIG. 93: Depiction of an embodiment of a cryopreserved TIL manufacturingprocess (22 days).

FIG. 94: Table of process improvements from Gen 1 to Gen 2.

FIG. 95A, FIG. 95B, and FIG. 95C: Total viable cells, growth rate, andviability. On Day 22 the volume reduced cell product is pooled andsampled to determine culture performance prior to wash and formulation.(A) Samples are analyzed on the NC-200 automated cell counter aspreviously described. Total viable cell density is determined by thegrand mean of duplicate counts from 4 independent samples. The Gen 2process yields a TIL product of similar dose to Gen 1 (Gen 1mean=4.10×10¹⁰±2.8×10¹⁰, Gen 2 mean=4.12×10¹⁰±2.5×10¹⁰. (B) The growthrate is calculated for the REP phase as gr=ln(N(t)/N(0))/t. (C) Cellviability was assessed from 9 process development lots using theCellometer K2 as previously described. No significant decrease in cellviability was observed following a single freeze-thaw cycle of theformulated product. Average reduction in viability upon thaw andsampling is 2.19%.

FIG. 96A, FIG. 96B, and FIG. 96C: Gen 2 products are highly pure T-cellcultures which express costimulatory molecules at levels comparable toGen 1. (A) Fresh formulated drug products were assayed for identity byflow cytometry for release. Gen 1 and Gen 2 processes produce highpurity T-cell cultures as defined by CD45+,CD3+(double positive)phenotype. (B & C) Cryopreserved satellite vials of formulated drugproduct were thawed and assayed for extended phenotype by flow cytometryas previously described. Gen 1 and Gen 2 products express similar levelsof costimulatory molecules CD27 and CD28 on T-cell subsets.Costimulatory molecules such as CD27 and CD28 are required to supplysecondary and tertiary signaling necessary for effector cellproliferation upon T-cell receptor engagement. P-value was calculatedusing Mann-Whitney ‘t’ test.

FIG. 97: Gen 2 products exhibit similar telomere lengths. However, someTIL populations may trend toward longer relative telomere.

FIG. 98: Gen 2 drug products secrete IFNγ in response to CD3, CD28, andCD137 engagement.

FIG. 99A and FIG. 99B: T-cell receptor diversity. (A) Unique CDR3sequences were indexed relative to frequency in each product to yield ascore representative of the overall diversity of T-cell receptors in theproduct. (B) The average total number of unique CDR3 sequences presentin each infusion product.

FIG. 100: An embodiment of a TIL manufacturing process of the presentinvention.

FIG. 101: Enhancement in expansion during the pre-REP withIL-2/IL-15/IL-21 in multiple tumor histologies.

FIG. 102A and FIG. 102B: IL-2/IL-15/IL-21 enhanced the percentage ofCD8+ cells in lung carcinoma, but not in melanoma. TIL derived from (A)melanoma (n=4), and (B) lung (n=7) were assessed phenotypically for CD4+and CD8+ cells using flow cytometry post pre-REP.

FIG. 103A and FIG. 103B: Expression of CD27 was slightly enhanced inCD8+ cells in cultures treated with IL-2/IL-15/IL-21. TIL derived from(A) melanoma (n=4), and (B) lung (n=7) were assessed phenotypically forCD27+ and CD28+ in the CD4+ and CD8+ cells using flow cytometry postpre-REP.

FIG. 104A and FIG. 104B: T cell subsets were unaltered with the additionof IL-15/IL-21. TIL were assessed phenotypically for effector/memorysubsets (CD45RA and CCR7) in the CD8+ and CD4+(data not shown) cellsfrom (A) melanoma (n=4), and (B) lung (n=8) via flow cytometry postpre-REP.

FIG. 105A, FIG. 105B, and FIG. 105C: Functional capacity of TIL wasdifferentially enhanced with IL-2/IL-15/IL-21. TIL derived from (A)melanoma (n=4) and (B) lung (n=5) were assessed for CD107a+ expressionin response to PMA stimulation for 4 hours in the CD4+ and CD8+ cells,by flow cytometry. (C) pre-REP TIL derived from melanoma and lung werestimulated for 24 hours with soluble anti-CD3 antibody and thesupernatants assessed for IFNγ by ELISA.

FIG. 106A and FIG. 106B: The TCRvβ repertoire (24 specificities) wereassessed in the TIL derived from a (A) melanoma and (B) lung tumor usingthe Beckman Coulter kit for flow cytometry.

FIG. 107: Scheme of Gen 2 cryopreserved LN-144 manufacturing process.

FIG. 108: Scheme of study design of multicenter phase 2 clinical trialof novel cryopreserved TILs administered to patients with metastaticmelanoma.

FIG. 109: Table illustrating the Comparison Patient Characteristics fromCohort 1 (ASCO 2017) vs Cohort 2.

FIG. 110: Table illustrating treatment emergent adverse events (≥30%).

FIG. 111: Efficacy of the infusion product and TIL therapy.

FIG. 112: Clinical status of response evaluable patients with SD or abetter response.

FIG. 113: Percent change in sum of diameters.

FIG. 114: An increase of HMGB1 level was observed upon TIL treatment.

FIG. 115: An increase in the biomarker IL-10 was observed post-LN-144infusion.

FIG. 116: Updated patient characteristics for Cohort 2 of the phase 2clinical trial in metastatic melanoma from the second data cut (N=17patients).

FIG. 117: Treatment emergent adverse events for Cohort 2 (≥30%) from thesecond data cut (N=17 patients).

FIG. 118: Time to response for evaluable patients (stable disease orbetter) in Cohort 2 from the second data cut (N=17 patients). Of the 10patients in the efficacy set, one patient (Patient 10) was not evaluabledue to a melanoma-related death prior to the first tumor assessment notrepresented on the figure.

FIG. 119: Updated efficacy data for Cohort 2 from the second data cut(N=17 patients). The mean number of TILs infused is 34×10⁹. The mediannumber of prior therapies was 4.5. Patients with a BRAF mutationresponded as well as patients with wild-type BRAF (a * refers topatients with a BRAF mutation). One patient (Patient 10) was notevaluable due to a melanoma-related death prior to the first tumorassessment but was still considered in the efficacy set. Abbreviations:PR, partial response; SD, stable disease; PD, progressive disease.

FIG. 120: Updated efficacy data for evaluable patients from Cohort 2from the second data cut (N=17 patients). The * indicates anon-evaluable patient that did not reach the first assessment. Allefficacy-evaluable patients had received prior anti-PD-1 and anti-CTLA-4checkpoint inhibitor therapies.

FIG. 121: Representative computed tomography scan of a patient (003-015)with a PR from Cohort 2, second data cut.

FIG. 122: Correlation of IFN-γ induction by TIL product prior toinfusion with clinical reduction in tumor size on Day 42 post TILinfusion.

FIG. 123: IP-10 (CXCL10) levels (pg/mL, log₁₀) pre- and post-infusion ofan embodiment of Gen 2 TIL product. IP-10 is a marker of cell adhesionand homing.

FIG. 124: IP-10 (CXCL10) levels (pg/mL, log₁₀) pre- and post-infusion ofan embodiment of Gen 1 TIL product.

FIG. 125: MCP-1 levels (pg/mL, log₁₀) pre- and post-infusion of anembodiment of Gen 2 TIL product. MCP-1 is a marker of cell adhesion andhoming.

FIG. 126: MCP-1 levels (pg/mL, log₁₀) pre- and post-infusion of anembodiment of Gen 1 TIL product.

FIG. 127: Data from Phase 2 studies in cervical carcinoma and head andneck squamous cell carcinoma (HNSCC). SD=stable disease. PR=progressivedisease. PR=partial response.

FIG. 128: Provides a chart showing the overview of the 3 phases of theexperiment, as discussed in Example 21.

FIG. 129: Calculate the amount of IL-2 required from final producttable.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO:1 is the amino acid sequence of the heavy chain of muromonab.

SEQ ID NO:2 is the amino acid sequence of the light chain of muromonab.

SEQ ID NO:3 is the amino acid sequence of a recombinant human IL-2protein.

SEQ ID NO:4 is the amino acid sequence of aldesleukin.

SEQ ID NO:5 is the amino acid sequence of a recombinant human IL-4protein.

SEQ ID NO:6 is the amino acid sequence of a recombinant human IL-7protein.

SEQ ID NO:7 is the amino acid sequence of a recombinant human IL-15protein.

SEQ ID NO:8 is the amino acid sequence of a recombinant human IL-21protein.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Adoptive cell therapy utilizing TILs cultured ex vivo by the RapidExpansion Protocol (REP) has produced successful adoptive cell therapyfollowing host immunosuppression in patients with melanoma. Currentinfusion acceptance parameters rely on readouts of the composition ofTILs (e.g., CD28, CD8, or CD4 positivity) and on the numerical folds ofexpansion and viability of the REP product.

Current REP protocols give little insight into the health of the TILthat will be infused into the patient. T cells undergo a profoundmetabolic shift during the course of their maturation from naïve toeffector T cells (see Chang, et al., Nat. Immunol. 2016, 17, 364, herebyexpressly incorporated in its entirety, and in particular for thediscussion and markers of anaerobic and aerobic metabolism). Forexample, naïve T cells rely on mitochondrial respiration to produce ATP,while mature, healthy effector T cells such as TIL are highlyglycolytic, relying on aerobic glycolysis to provide the bioenergeticssubstrates they require for proliferation, migration, activation, andanti-tumor efficacy.

Previous papers report that limiting glycolysis and promotingmitochondrial metabolism in TILs prior to transfer is desirable as cellsthat are relying heavily on glycolysis will suffer nutrient deprivationupon adoptive transfer which results in a majority of the transferredcells dying. Thus, the art teaches that promoting mitochondrialmetabolism might promote in vivo longevity and in fact suggests usinginhibitors of glycolysis before induction of the immune response. SeeChang et al. (Chang, et al., Nat. Immunol. 2016, 17(364).

The present invention is further directed in some embodiments to methodsfor evaluating and quantifying this increase in metabolic health. Thus,the present invention provides methods of assaying the relative healthof a TIL population using one or more general evaluations of metabolism,including, but not limited to, rates and amounts of glycolysis,oxidative phosphorylation, spare respiratory capacity (SRC), andglycolytic reserve.

Furthermore, the present invention is further directed in someembodiments to methods for evaluating and quantifying this increase inmetabolic health. Thus, the present invention provides methods ofassaying the relative health of a TIL population using one or moregeneral evaluations of metabolism, including, but not limited to, ratesand amounts of glycolysis, oxidative phosphorylation, spare respiratorycapacity (SRC), and glycolytic reserve.

In addition, optional additional evaluations include, but are notlimited to, ATP production, mitochondrial mass and glucose uptake.

II. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents and publicationsreferred to herein are incorporated by reference in their entireties.

The term “in vivo” refers to an event that takes place in a subject'sbody.

The term “in vitro” refers to an event that takes places outside of asubject's body. In vitro assays encompass cell-based assays in whichcells alive or dead are employed and may also encompass a cell-freeassay in which no intact cells are employed.

The term “ex vivo” refers to an event which involves treating orperforming a procedure on a cell, tissue and/or organ which has beenremoved from a subject's body. Aptly, the cell, tissue and/or organ maybe returned to the subject's body in a method of surgery or treatment.

The term “rapid expansion” means an increase in the number ofantigen-specific TILs of at least about 3-fold (or 4-, 5-, 6-, 7-, 8-,or 9-fold) over a period of a week, more preferably at least about10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold) over a periodof a week, or most preferably at least about 100-fold over a period of aweek. A number of rapid expansion protocols are outlined below.

By “tumor infiltrating lymphocytes” or “TILs” herein is meant apopulation of cells originally obtained as white blood cells that haveleft the bloodstream of a subject and migrated into a tumor. TILsinclude, but are not limited to, CD8⁺ cytotoxic T cells (lymphocytes),Th1 and Th17 CD4⁺ T cells, natural killer cells, dendritic cells and M1macrophages. TILs include both primary and secondary TILs. “PrimaryTILs” are those that are obtained from patient tissue samples asoutlined herein (sometimes referred to as “freshly harvested”), and“secondary TILs” are any TIL cell populations that have been expanded orproliferated as discussed herein, including, but not limited to bulkTILs and expanded TILs (“REP TILs” or “post-REP TILs”). TIL cellpopulations can include genetically modified TILs.

By “population of cells” (including TILs) herein is meant a number ofcells that share common traits. In general, populations generally rangefrom 1×10⁶ to 1×10¹⁰ in number, with different TIL populationscomprising different numbers. For example, initial growth of primaryTILs in the presence of IL-2 results in a population of bulk TILs ofroughly 1×10⁸ cells. REP expansion is generally done to providepopulations of 1.5×10⁹ to 1.5×10¹⁰ cells for infusion.

By “cryopreserved TILs” herein is meant that TILs, either primary, bulk,or expanded (REP TILs), are treated and stored in the range of about−150° C. to −60° C. General methods for cryopreservation are alsodescribed elsewhere herein, including in the Examples. For clarity,“cryopreserved TILs” are distinguishable from frozen tissue sampleswhich may be used as a source of primary TILs.

By “thawed cryopreserved TILs” herein is meant a population of TILs thatwas previously cryopreserved and then treated to return to roomtemperature or higher, including but not limited to cell culturetemperatures or temperatures wherein TILs may be administered to apatient.

TILs can generally be defined either biochemically, using cell surfacemarkers, or functionally, by their ability to infiltrate tumors andeffect treatment. TILs can be generally categorized by expressing one ormore of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56,CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TILscan be functionally defined by their ability to infiltrate solid tumorsupon reintroduction into a patient.

The term “cryopreservation media” or “cryopreservation medium” refers toany medium that can be used for cryopreservation of cells. Such mediacan include media comprising 7% to 10% DMSO. Exemplary media includeCryoStor CS10, Hyperthermasol, as well as combinations thereof. The term“CS10” refers to a cryopreservation medium which is obtained fromStemcell Technologies or from Biolife Solutions. The CS10 medium may bereferred to by the trade name “CryoStor® CS10”. The CS10 medium is aserum-free, animal component-free medium which comprises DMSO.

The term “central memory T cell” refers to a subset of T cells that inthe human are CD45R0+ and constitutively express CCR7 (CCR7^(hi)) andCD62L (CD62^(hi)). The surface phenotype of central memory T cells alsoincludes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors forcentral memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. Centralmemory T cells primarily secret IL-2 and CD40L as effector moleculesafter TCR triggering. Central memory T cells are predominant in the CD4compartment in blood, and in the human are proportionally enriched inlymph nodes and tonsils.

The term “effector memory T cell” refers to a subset of human ormammalian T cells that, like central memory T cells, are CD45R0+, buthave lost the constitutive expression of CCR7 (CCR7^(lo)) and areheterogeneous or low for CD62L expression) (CD62L^(lo). The surfacephenotype of central memory T cells also includes TCR, CD3, CD127(IL-7R), and IL-15R. Transcription factors for central memory T cellsinclude BLIMP1. Effector memory T cells rapidly secret high levels ofinflammatory cytokines following antigenic stimulation, includinginterferon-γ, IL-4, and IL-5. Effector memory T cells are predominant inthe CD8 compartment in blood, and in the human are proportionallyenriched in the lung, liver, and gut. CD8+ effector memory T cells carrylarge amounts of perforin.

The term “closed system” refers to a system that is closed to theoutside environment. Any closed system appropriate for cell culturemethods can be employed with the methods of the present invention.Closed systems include, for example, but are not limited to closedG-containers. Once a tumor segment is added to the closed system, thesystem is no opened to the outside environment until the TILs are readyto be administered to the patient.

The terms “fragmenting,” “fragment,” and “fragmented,” as used herein todescribe processes for disrupting a tumor, includes mechanicalfragmentation methods such as crushing, slicing, dividing, andmorcellating tumor tissue as well as any other method for disrupting thephysical structure of tumor tissue.

The terms “peripheral blood mononuclear cells” and “PBMCs” refers to aperipheral blood cell having a round nucleus, including lymphocytes (Tcells, B cells, NK cells) and monocytes. Preferably, the peripheralblood mononuclear cells are irradiated allogeneic peripheral bloodmononuclear cells. PBMCs are a type of antigen-presenting cell.

The term “anti-CD3 antibody” refers to an antibody or variant thereof,e.g., a monoclonal antibody and including human, humanized, chimeric ormurine antibodies which are directed against the CD3 receptor in the Tcell antigen receptor of mature T cells. Anti-CD3 antibodies includeOKT-3, also known as muromonab. Anti-CD3 antibodies also include theUHCT1 clone, also known as T3 and CD3ε. Other anti-CD3 antibodiesinclude, for example, otelixizumab, teplizumab, and visilizumab.

The term “OKT-3” (also referred to herein as “OKT3”) refers to amonoclonal antibody or biosimilar or variant thereof, including human,humanized, chimeric, or murine antibodies, directed against the CD3receptor in the T cell antigen receptor of mature T cells, and includescommercially-available forms such as OKT-3 (30 ng/mL, MACS GMP CD3 pure,Miltenyi Biotech, Inc., San Diego, Calif., USA) and muromonab orvariants, conservative amino acid substitutions, glycoforms, orbiosimilars thereof. The amino acid sequences of the heavy and lightchains of muromonab are given in Table 1 (SEQ ID NO:1 and SEQ ID NO:2).A hybridoma capable of producing OKT-3 is deposited with the AmericanType Culture Collection and assigned the ATCC accession number CRL 8001.A hybridoma capable of producing OKT-3 is also deposited with EuropeanCollection of Authenticated Cell Cultures (ECACC) and assigned CatalogueNo. 86022706.

TABLE 1 Amino acid sequences of muromonab. IdentifierSequence (One-Letter Amino Acid Symbols) SEQ ID NO: 1QVQLQQSGAE LARPGASVKM SCKASGYTFT RYTMHWVKQR PGQGLEWIGY INPSRGYTNY  60Muromonab heavyNQKFKDKATL TTDKSSSTAY MQLSSLTSED SAVYYCARYY DDHYCLDYWG QGTTLTVSSA 120chain KTTAPSVYPL APVCGGTTGS SVTLGCLVKG YFPEPVTLTW NSGSLSSGVH TFPAVLQSDL180 YTLSSSVTVT SSTWPSQSIT CNVAHPASST KVDKKIEPRP KSCDKTHTCP PCPAPELLGG240 PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN300 STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE360 LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW420 QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 450 SEQ ID NO: 2QIVLTQSPAI MSASPGEKVT MTCSASSSVS YMNWYQQKSG TSPKRWIYDT SKLASGVPAH  60Muromonab lightFRGSGSGTSY SLTISGMEAE DAATYYCQQW SSNPFTFGSG TKLEINRADT APTVSIFPPS 120chain SEQLTSGGAS VVCFLNNFYP KDINVKWKID GSERQNGVLN SWTDQDSKDS TYSMSSTLTL180 TKDEYERHNS YTCEATHKTS TSPIVKSFNR NEC 213

The term “IL-2” (also referred to herein as “IL2”) refers to the T cellgrowth factor known as interleukin-2, and includes all forms of IL-2including human and mammalian forms, conservative amino acidsubstitutions, glycoforms, biosimilars, and variants thereof. IL-2 isdescribed, e.g., in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek,Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which areincorporated by reference herein. The amino acid sequence of recombinanthuman IL-2 suitable for use in the invention is given in Table 2 (SEQ IDNO:3). For example, the term IL-2 encompasses human, recombinant formsof IL-2 such as aldesleukin (PROLEUKIN, available commercially frommultiple suppliers in 22 million IU per single use vials), as well asthe form of recombinant IL-2 commercially supplied by CellGenix, Inc.,Portsmouth, N.H., USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd.,East Brunswick, N.J., USA (Cat. No. CYT-209-b) and other commercialequivalents from other vendors. Aldesleukin (des-alanyl-1, serine-125human IL-2) is a nonglycosylated human recombinant form of IL-2 with amolecular weight of approximately 15 kDa. The amino acid sequence ofaldesleukin suitable for use in the invention is given in Table 2 (SEQID NO:4). The term IL-2 also encompasses pegylated forms of IL-2, asdescribed herein, including the pegylated IL2 prodrug NKTR-214,available from Nektar Therapeutics, South San Francisco, Calif., USA.NKTR-214 and pegylated IL-2 suitable for use in the invention isdescribed in U.S. Patent Application Publication No. US 2014/0328791 A1and International Patent Application Publication No. WO 2012/065086 A1,the disclosures of which are incorporated by reference herein.Alternative forms of conjugated IL-2 suitable for use in the inventionare described in U.S. Pat. Nos. 4,766,106, 5,206,344, 5,089,261 and4,902,502, the disclosures of which are incorporated by referenceherein. Formulations of IL-2 suitable for use in the invention aredescribed in U.S. Pat. No. 6,706,289, the disclosure of which isincorporated by reference herein.

TABLE 2 Amino acid sequences of interleukins. IdentifierSequence (One-Letter Amino Acid Symbols) SEQ ID NO: 3MAPTSSSTKK TQLQLEHLLL DLQMILNGIN NYKNPKLTRM LTFKFYMPKK ATELKHLQCL  60recombinantEEELKPLEEV LNLAQSKNFH LRPRDLISNI NVIVLELKGS ETTFMCEYAD ETATIVEFLN 120human IL-2 RWITFCQSII STLT 134 (rhIL-2) SEQ ID NO: 4PTSSSTKKTQ LQLEHLLLDL QMILNGINNY KNPKLTRMLT FKFYMPKKAT ELKHLQCLEE  60AldesleukinELKPLEEVLN LAQSKNFHLR PRDLISNINV IVLELKGSET TFMCEYADET ATIVEFLNRW 120ITFSQSIIST LT 132 SEQ ID NO: 5MHKCDITLQE IIKTLNSLTE QKTLCTELTV TDIFAASKNT TEKETFCRAA TVLRQFYSHH  60recombinantEKDTRCLGAT AQQFHRHKQL IRFLKRLDRN LWGLAGLNSC PVKEANQSTL ENFLERLKTI 120human IL-4 MREKYSKCSS 130 (rhIL-4) SEQ ID NO: 6MDCDIEGKDG KQYESVLMVS IDQLLDSMKE IGSNCLNNEF NFFKRHICDA NKEGMFLFRA  60recombinantARKLRQFLKM NSTGDFDLHL LKVSEGTTIL LNCTGQVKGR KPAALGEAQP TKSLEENKSL 120human IL-7 KEQKKLNDLC FLKRLLQEIK TCWNKILMGT KEH 153 (rhIL-7)SEQ ID NO: 7MNWVNVISDL KKIEDLIQSM HIDATLYTES DVHPSCKVTA MKCFLLELQV ISLESGDASI  60recombinant HDTVENLIIL ANNSLSSNGN VTESGCKECE ELEEKNIKEF LQSFVHIVQM FINTS115 human IL-15 (rhIL-15) SEQ ID NO: 8MQDRHMIRMR QLIDIVDQLK NYVNDLVPEF LPAPEDVETN CEWSAFSCFQ KAQLKSANTG  60recombinantNNERIINVSI KKLKRKPPST NAGRRQKHRL TCPSCDSYEK KPPKEFLERF KSLLQKMIHQ 120human IL-21 HLSSRTHGSE DS 132 (rhIL-21)

The term “IL-4” (also referred to herein as “IL4”) refers to thecytokine known as interleukin 4, which is produced by Th2 T cells and byeosinophils, basophils, and mast cells. IL-4 regulates thedifferentiation of naïve helper T cells (Th0 cells) to Th2 T cells.Steinke and Borish, Respir. Res. 2001, 2, 66-70. Upon activation byIL-4, Th2 T cells subsequently produce additional IL-4 in a positivefeedback loop. IL-4 also stimulates B cell proliferation and class IIMHC expression, and induces class switching to IgE and IgG₁ expressionfrom B cells. Recombinant human IL-4 suitable for use in the inventionis commercially available from multiple suppliers, includingProSpec-Tany TechnoGene Ltd., East Brunswick, N.J., USA (Cat. No.CYT-211) and ThermoFisher Scientific, Inc., Waltham, Mass., USA (humanIL-15 recombinant protein, Cat. No. Gibco CTP0043). The amino acidsequence of recombinant human IL-4 suitable for use in the invention isgiven in Table 2 (SEQ ID NO:5).

The term “IL-7” (also referred to herein as “IL7”) refers to aglycosylated tissue-derived cytokine known as interleukin 7, which maybe obtained from stromal and epithelial cells, as well as from dendriticcells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate thedevelopment of T cells. IL-7 binds to the IL-7 receptor, a heterodimerconsisting of IL-7 receptor alpha and common gamma chain receptor, whichin a series of signals important for T cell development within thethymus and survival within the periphery. Recombinant human IL-7suitable for use in the invention is commercially available frommultiple suppliers, including ProSpec-Tany TechnoGene Ltd., EastBrunswick, N.J., USA (Cat. No. CYT-254) and ThermoFisher Scientific,Inc., Waltham, Mass., USA (human IL-15 recombinant protein, Cat. No.Gibco PHC0071). The amino acid sequence of recombinant human IL-7suitable for use in the invention is given in Table 2 (SEQ ID NO:6).

The term “IL-15” (also referred to herein as “IL15”) refers to the Tcell growth factor known as interleukin-15, and includes all forms ofIL-2 including human and mammalian forms, conservative amino acidsubstitutions, glycoforms, biosimilars, and variants thereof. IL-15 isdescribed, e.g., in Fehniger and Caligiuri, Blood 2001, 97, 14-32, thedisclosure of which is incorporated by reference herein. IL-15 shares βand γ signaling receptor subunits with IL-2. Recombinant human IL-15 isa single, non-glycosylated polypeptide chain containing 114 amino acids(and an N-terminal methionine) with a molecular mass of 12.8 kDa.Recombinant human IL-15 is commercially available from multiplesuppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, N.J.,USA (Cat. No. CYT-230-b) and ThermoFisher Scientific, Inc., Waltham,Mass., USA (human IL-15 recombinant protein, Cat. No. 34-8159-82). Theamino acid sequence of recombinant human IL-15 suitable for use in theinvention is given in Table 2 (SEQ ID NO:7).

The term “IL-21” (also referred to herein as “IL21”) refers to thepleiotropic cytokine protein known as interleukin-21, and includes allforms of IL-21 including human and mammalian forms, conservative aminoacid substitutions, glycoforms, biosimilars, and variants thereof. IL-21is described, e.g., in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014,13, 379-95, the disclosure of which is incorporated by reference herein.IL-21 is primarily produced by natural killer T cells and activatedhuman CD4⁺ T cells. Recombinant human IL-21 is a single,non-glycosylated polypeptide chain containing 132 amino acids with amolecular mass of 15.4 kDa. Recombinant human IL-21 is commerciallyavailable from multiple suppliers, including ProSpec-Tany TechnoGeneLtd., East Brunswick, N.J., USA (Cat. No. CYT-408-b) and ThermoFisherScientific, Inc., Waltham, Mass., USA (human IL-21 recombinant protein,Cat. No. 14-8219-80). The amino acid sequence of recombinant human IL-21suitable for use in the invention is given in Table 2 (SEQ ID NO:8).

When “an anti-tumor effective amount”, “an tumor-inhibiting effectiveamount”, or “therapeutic amount” is indicated, the precise amount of thecompositions of the present invention to be administered can bedetermined by a physician with consideration of individual differencesin age, weight, tumor size, extent of infection or metastasis, andcondition of the patient (subject). It can generally be stated that apharmaceutical composition comprising the tumor infiltrating lymphocytes(e.g. secondary TILs or genetically modified cytotoxic lymphocytes)described herein may be administered at a dosage of 10⁴ to 10¹¹ cells/kgbody weight (e.g., 10⁵ to 10⁶, 10⁵ to 10¹⁰, 10⁵ to 10¹¹, 10⁶ to 10¹⁰,10⁶ to 10¹¹, 10⁷ to 10¹¹, 10⁷ to 10¹⁰, 10⁸ to 10¹¹, 10⁸ to 10¹⁰, 10⁹ to10¹¹, or 10⁹ to 10¹⁰ cells/kg body weight), including all integer valueswithin those ranges. Tumor infiltrating lymphocytes (including in somecases, genetically modified cytotoxic lymphocytes) compositions may alsobe administered multiple times at these dosages. The tumor infiltratinglymphocytes (including in some cases, genetically) can be administeredby using infusion techniques that are commonly known in immunotherapy(see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). Theoptimal dosage and treatment regime for a particular patient can readilybe determined by one skilled in the art of medicine by monitoring thepatient for signs of disease and adjusting the treatment accordingly.

The term “hematological malignancy” refers to mammalian cancers andtumors of the hematopoietic and lymphoid tissues, including but notlimited to tissues of the blood, bone marrow, lymph nodes, and lymphaticsystem. Hematological malignancies are also referred to as “liquidtumors.” Hematological malignancies include, but are not limited to,acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL),small lymphocytic lymphoma (SLL), acute myelogenous leukemia (AML),chronic myelogenous leukemia (CIVIL), acute monocytic leukemia (AMoL),Hodgkin's lymphoma, and non-Hodgkin's lymphomas. The term “B cellhematological malignancy” refers to hematological malignancies thataffect B cells.

The term “solid tumor” refers to an abnormal mass of tissue that usuallydoes not contain cysts or liquid areas. Solid tumors may be benign ormalignant. The term “solid tumor cancer refers to malignant, neoplastic,or cancerous solid tumors. Solid tumor cancers include, but are notlimited to, sarcomas, carcinomas, and lymphomas, such as cancers of thelung, breast, prostate, colon, rectum, and bladder. The tissue structureof solid tumors includes interdependent tissue compartments includingthe parenchyma (cancer cells) and the supporting stromal cells in whichthe cancer cells are dispersed and which may provide a supportingmicroenvironment.

The term “liquid tumor” refers to an abnormal mass of cells that isfluid in nature. Liquid tumor cancers include, but are not limited to,leukemias, myelomas, and lymphomas, as well as other hematologicalmalignancies. TILs obtained from liquid tumors may also be referred toherein as marrow infiltrating lymphocytes (MILs).

The term “microenvironment,” as used herein, may refer to the solid orhematological tumor microenvironment as a whole or to an individualsubset of cells within the microenvironment. The tumor microenvironment,as used herein, refers to a complex mixture of “cells, soluble factors,signaling molecules, extracellular matrices, and mechanical cues thatpromote neoplastic transformation, support tumor growth and invasion,protect the tumor from host immunity, foster therapeutic resistance, andprovide niches for dominant metastases to thrive,” as described inSwartz, et al., Cancer Res., 2012, 72, 2473. Although tumors expressantigens that should be recognized by T cells, tumor clearance by theimmune system is rare because of immune suppression by themicroenvironment.

In an embodiment, the invention includes a method of treating a cancerwith a population of TILs, wherein a patient is pre-treated withnon-myeloablative chemotherapy prior to an infusion of TILs according tothe invention. In some embodiments, the population of TILs may beprovided wherein a patient is pre-treated with nonmyeloablativechemotherapy prior to an infusion of TILs according to the presentinvention. In an embodiment, the non-myeloablative chemotherapy iscyclophosphamide 60 mg/kg/d for 2 days (days 27 and 26 prior to TILinfusion) and fludarabine 25 mg/m2/d for 5 days (days 27 to 23 prior toTIL infusion). In an embodiment, after non-myeloablative chemotherapyand TIL infusion (at day 0) according to the invention, the patientreceives an intravenous infusion of IL-2 intravenously at 720,000 IU/kgevery 8 hours to physiologic tolerance.

Experimental findings indicate that lymphodepletion prior to adoptivetransfer of tumor-specific T lymphocytes plays a key role in enhancingtreatment efficacy by eliminating regulatory T cells and competingelements of the immune system (“cytokine sinks”). Accordingly, someembodiments of the invention utilize a lymphodepletion step (sometimesalso referred to as “immunosuppressive conditioning”) on the patientprior to the introduction of the rTILs of the invention.

The terms “co-administration,” “co-administering,” “administered incombination with,” “administering in combination with,” “simultaneous,”and “concurrent,” as used herein, encompass administration of two ormore active pharmaceutical ingredients (in a preferred embodiment of thepresent invention, for example, at least one potassium channel agonistin combination with a plurality of TILs) to a subject so that bothactive pharmaceutical ingredients and/or their metabolites are presentin the subject at the same time. Co-administration includes simultaneousadministration in separate compositions, administration at differenttimes in separate compositions, or administration in a composition inwhich two or more active pharmaceutical ingredients are present.Simultaneous administration in separate compositions and administrationin a composition in which both agents are present are preferred.

The term “effective amount” or “therapeutically effective amount” refersto that amount of a compound or combination of compounds as describedherein that is sufficient to effect the intended application including,but not limited to, disease treatment. A therapeutically effectiveamount may vary depending upon the intended application (in vitro or invivo), or the subject and disease condition being treated (e.g., theweight, age and gender of the subject), the severity of the diseasecondition, or the manner of administration. The term also applies to adose that will induce a particular response in target cells (e.g., thereduction of platelet adhesion and/or cell migration). The specific dosewill vary depending on the particular compounds chosen, the dosingregimen to be followed, whether the compound is administered incombination with other compounds, timing of administration, the tissueto which it is administered, and the physical delivery system in whichthe compound is carried.

The terms “treatment”, “treating”, “treat”, and the like, refer toobtaining a desired pharmacologic and/or physiologic effect. The effectmay be prophylactic in terms of completely or partially preventing adisease or symptom thereof and/or may be therapeutic in terms of apartial or complete cure for a disease and/or adverse effectattributable to the disease. “Treatment”, as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease but has not yet been diagnosed ashaving it; (b) inhibiting the disease, i.e., arresting its developmentor progression; and (c) relieving the disease, i.e., causing regressionof the disease and/or relieving one or more disease symptoms.“Treatment” is also meant to encompass delivery of an agent in order toprovide for a pharmacologic effect, even in the absence of a disease orcondition. For example, “treatment” encompasses delivery of acomposition that can elicit an immune response or confer immunity in theabsence of a disease condition, e.g., in the case of a vaccine.

The term “heterologous” when used with reference to portions of anucleic acid or protein indicates that the nucleic acid or proteincomprises two or more subsequences that are not found in the samerelationship to each other in nature. For instance, the nucleic acid istypically recombinantly produced, having two or more sequences fromunrelated genes arranged to make a new functional nucleic acid, e.g., apromoter from one source and a coding region from another source, orcoding regions from different sources. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

The terms “sequence identity,” “percent identity,” and “sequence percentidentity” (or synonyms thereof, e.g., “99% identical”) in the context oftwo or more nucleic acids or polypeptides, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of nucleotides or amino acid residues that are the same, whencompared and aligned (introducing gaps, if necessary) for maximumcorrespondence, not considering any conservative amino acidsubstitutions as part of the sequence identity. The percent identity canbe measured using sequence comparison software or algorithms or byvisual inspection. Various algorithms and software are known in the artthat can be used to obtain alignments of amino acid or nucleotidesequences. Suitable programs to determine percent sequence identityinclude for example the BLAST suite of programs available from the U.S.Government's National Center for Biotechnology Information BLAST website. Comparisons between two sequences can be carried using either theBLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acidsequences, while BLASTP is used to compare amino acid sequences. ALIGN,ALIGN-2 (Genentech, South San Francisco, Calif.) or MegAlign, availablefrom DNASTAR, are additional publicly available software programs thatcan be used to align sequences. One skilled in the art can determineappropriate parameters for maximal alignment by particular alignmentsoftware. In certain embodiments, the default parameters of thealignment software are used.

As used herein, the term “variant” encompasses but is not limited toantibodies or fusion proteins which comprise an amino acid sequencewhich differs from the amino acid sequence of a reference antibody byway of one or more substitutions, deletions and/or additions at certainpositions within or adjacent to the amino acid sequence of the referenceantibody. The variant may comprise one or more conservativesubstitutions in its amino acid sequence as compared to the amino acidsequence of a reference antibody. Conservative substitutions mayinvolve, e.g., the substitution of similarly charged or uncharged aminoacids. The variant retains the ability to specifically bind to theantigen of the reference antibody. The term variant also includespegylated antibodies or proteins.

By “tumor infiltrating lymphocytes” or “TILs” herein is meant apopulation of cells originally obtained as white blood cells that haveleft the bloodstream of a subject and migrated into a tumor. TILsinclude, but are not limited to, CD8⁺ cytotoxic T cells (lymphocytes),Th1 and Th17 CD4⁺ T cells, natural killer cells, dendritic cells and M1macrophages. TILs include both primary and secondary TILs. “PrimaryTILs” are those that are obtained from patient tissue samples asoutlined herein (sometimes referred to as “freshly harvested”), and“secondary TILs” are any TIL cell populations that have been expanded orproliferated as discussed herein, including, but not limited to bulkTILs, expanded TILs (“REP TILs”) as well as “reREP TILs” as discussedherein. reREP TILs can include for example second expansion TILs orsecond additional expansion TILs (such as, for example, those describedin Step D of FIG. 27, including TILs referred to as reREP TILs).

TILs can generally be defined either biochemically, using cell surfacemarkers, or functionally, by their ability to infiltrate tumors andeffect treatment. TILs can be generally categorized by expressing one ormore of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56,CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally, and alternatively,TILs can be functionally defined by their ability to infiltrate solidtumors upon reintroduction into a patient. TILS may further becharacterized by potency—for example, TILS may be considered potent if,for example, interferon (IFN) release is greater than about 50 pg/mL,greater than about 100 pg/mL, greater than about 150 pg/mL, or greaterthan about 200 pg/mL.

The terms “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” are intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and inert ingredients. The useof such pharmaceutically acceptable carriers or pharmaceuticallyacceptable excipients for active pharmaceutical ingredients is wellknown in the art. Except insofar as any conventional pharmaceuticallyacceptable carrier or pharmaceutically acceptable excipient isincompatible with the active pharmaceutical ingredient, its use in thetherapeutic compositions of the invention is contemplated. Additionalactive pharmaceutical ingredients, such as other drugs, can also beincorporated into the described compositions and methods.

The terms “about” and “approximately” mean within a statisticallymeaningful range of a value. Such a range can be within an order ofmagnitude, preferably within 50%, more preferably within 20%, morepreferably still within 10%, and even more preferably within 5% of agiven value or range. The allowable variation encompassed by the terms“about” or “approximately” depends on the particular system under study,and can be readily appreciated by one of ordinary skill in the art.Moreover, as used herein, the terms “about” and “approximately” meanthat dimensions, sizes, formulations, parameters, shapes and otherquantities and characteristics are not and need not be exact, but may beapproximate and/or larger or smaller, as desired, reflecting tolerances,conversion factors, rounding off, measurement error and the like, andother factors known to those of skill in the art. In general, adimension, size, formulation, parameter, shape or other quantity orcharacteristic is “about” or “approximate” whether or not expresslystated to be such. It is noted that embodiments of very different sizes,shapes and dimensions may employ the described arrangements.

The transitional terms “comprising,” “consisting essentially of,” and“consisting of,” when used in the appended claims, in original andamended form, define the claim scope with respect to what unrecitedadditional claim elements or steps, if any, are excluded from the scopeof the claim(s). The term “comprising” is intended to be inclusive oropen-ended and does not exclude any additional, unrecited element,method, step or material. The term “consisting of” excludes any element,step or material other than those specified in the claim and, in thelatter instance, impurities ordinary associated with the specifiedmaterial(s). The term “consisting essentially of” limits the scope of aclaim to the specified elements, steps or material(s) and those that donot materially affect the basic and novel characteristic(s) of theclaimed invention. All compositions, methods, and kits described hereinthat embody the present invention can, in alternate embodiments, be morespecifically defined by any of the transitional terms “comprising,”“consisting essentially of,” and “consisting of.”

III. TIL Manufacturing Processes

An exemplary TIL process known as process 2A containing some of thesefeatures is depicted in FIG. 1, and some of the advantages of thisembodiment of the present invention over process 1C are described inFIG. 2, as does FIG. 84. Process 1C is shown for comparison in FIG. 3.Two alternative timelines for TIL therapy based on process 2A are shownin FIG. 4 (higher cell counts) and FIG. 5 (lower cell counts). Anembodiment of process 2A is shown in FIG. 6 as well as FIG. 27. FIGS. 83and 84 further provides an exemplary 2A process compared to an exemplary1C process.

As discussed herein, the present invention can include a step relatingto the restimulation of cryopreserved TILs to increase their metabolicactivity and thus relative health prior to transplant into a patient,and methods of testing said metabolic health. As generally outlinedherein, TILs are generally taken from a patient sample and manipulatedto expand their number prior to transplant into a patient. In someembodiments, the TILs may be optionally genetically manipulated asdiscussed below.

In some embodiments, the TILs may be cryopreserved. Once thawed, theymay also be restimulated to increase their metabolism prior to infusioninto a patient.

In some embodiments, the first expansion (including processes referredto as the preREP as well as processes shown in FIG. 27 as Step A) isshortened to 3 to 14 days and the second expansion (including processesreferred to as the REP as well as processes shown in FIG. 27 as Step B)is shorted to 7 to 14 days, as discussed in detail below as well as inthe examples and figures. In some embodiments, the first expansion (forexample, an expansion described as Step B in FIG. 27) is shortened to 11days and the second expansion (for example, an expansion as described inStep D in FIG. 27) is shortened to 11 days, as discussed in the Examplesand shown in FIGS. 4, 5 and 27. In some embodiments, the combination ofthe first expansion and second expansion (for example, expansionsdescribed as Step B and Step D in FIG. 27) is shortened to 22 days, asdiscussed in detail below and in the examples and figures.

The “Step” Designations A, B, C, etc., below are in reference to FIG. 27and in reference to certain embodiments described herein. The orderingof the Steps below and in FIG. 27 is exemplary and any combination ororder of steps, as well as additional steps, repetition of steps, and/oromission of steps is contemplated by the present application and themethods disclosed herein.

A. Step A: Obtain Patient Tumor Sample

In general, TILs are initially obtained from a patient tumor sample(“primary TILs”) and then expanded into a larger population for furthermanipulation as described herein, optionally cryopreserved, restimulatedas outlined herein and optionally evaluated for phenotype and metabolicparameters as an indication of TIL health.

A patient tumor sample may be obtained using methods known in the art,generally via surgical resection, needle biopsy or other means forobtaining a sample that contains a mixture of tumor and TIL cells. Ingeneral, the tumor sample may be from any solid tumor, including primarytumors, invasive tumors or metastatic tumors. The tumor sample may alsobe a liquid tumor, such as a tumor obtained from a hematologicalmalignancy. The solid tumor may be of any cancer type, including, butnot limited to, breast, pancreatic, prostate, colorectal, lung, brain,renal, stomach, and skin (including but not limited to squamous cellcarcinoma, basal cell carcinoma, and melanoma). In some embodiments,useful TILs are obtained from malignant melanoma tumors, as these havebeen reported to have particularly high levels of TILs.

The term “solid tumor” refers to an abnormal mass of tissue that usuallydoes not contain cysts or liquid areas. Solid tumors may be benign ormalignant. The term “solid tumor cancer” refers to malignant,neoplastic, or cancerous solid tumors. Solid tumor cancers include, butare not limited to, sarcomas, carcinomas, and lymphomas, such as cancersof the lung, breast, triple negative breast cancer, prostate, colon,rectum, and bladder. In some embodiments, the cancer is selected fromcervical cancer, head and neck cancer (including, for example, head andneck squamous cell carcinoma (HNSCC)) glioblastoma, ovarian cancer,sarcoma, pancreatic cancer, bladder cancer, breast cancer, triplenegative breast cancer, and non-small cell lung carcinoma. The tissuestructure of solid tumors includes interdependent tissue compartmentsincluding the parenchyma (cancer cells) and the supporting stromal cellsin which the cancer cells are dispersed and which may provide asupporting microenvironment.

The term “hematological malignancy” refers to mammalian cancers andtumors of the hematopoietic and lymphoid tissues, including but notlimited to tissues of the blood, bone marrow, lymph nodes, and lymphaticsystem. Hematological malignancies are also referred to as “liquidtumors.” Hematological malignancies include, but are not limited to,acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL),small lymphocytic lymphoma (SLL), acute myelogenous leukemia (AML),chronic myelogenous leukemia (CIVIL), acute monocytic leukemia (AMoL),Hodgkin's lymphoma, and non-Hodgkin's lymphomas. The term “B cellhematological malignancy” refers to hematological malignancies thataffect B cells.

Once obtained, the tumor sample is generally fragmented using sharpdissection into small pieces of between 1 to about 8 mm³, with fromabout 2-3 mm³ being particularly useful. The TILs are cultured fromthese fragments using enzymatic tumor digests. Such tumor digests may beproduced by incubation in enzymatic media (e.g., Roswell Park MemorialInstitute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg/mL gentamicine, 30units/mL of DNase and 1.0 mg/mL of collagenase) followed by mechanicaldissociation (e.g., using a tissue dissociator). Tumor digests may beproduced by placing the tumor in enzymatic media and mechanicallydissociating the tumor for approximately 1 minute, followed byincubation for 30 minutes at 37° C. in 5% CO₂, followed by repeatedcycles of mechanical dissociation and incubation under the foregoingconditions until only small tissue pieces are present. At the end ofthis process, if the cell suspension contains a large number of redblood cells or dead cells, a density gradient separation using FICOLLbranched hydrophilic polysaccharide may be performed to remove thesecells. Alternative methods known in the art may be used, such as thosedescribed in U.S. Patent Application Publication No. 2012/0244133 A1,the disclosure of which is incorporated by reference herein. Any of theforegoing methods may be used in any of the embodiments described hereinfor methods of expanding TILs or methods treating a cancer.

In general, the harvested cell suspension is called a “primary cellpopulation” or a “freshly harvested” cell population.

In some embodiments, fragmentation includes physical fragmentation,including for example, dissection as well as digestion. In someembodiments, the fragmentation is physical fragmentation. In someembodiments, the fragmentation is dissection. In some embodiments, thefragmentation is by digestion. In some embodiments, TILs can beinitially cultured from enzymatic tumor digests and tumor fragmentsobtained from patients. In an embodiment, TILs can be initially culturedfrom enzymatic tumor digests and tumor fragments obtained from patients.

In some embodiments, where the tumor is a solid tumor, the tumorundergoes physical fragmentation after the tumor sample is obtained in,for example, Step A (as provided in FIG. 27). In some embodiments, thefragmentation occurs before cryopreservation. In some embodiments, thefragmentation occurs after cryopreservation. In some embodiments, thefragmentation occurs after obtaining the tumor and in the absence of anycryopreservation. In some embodiments, the tumor is fragmented and 10,20, 30, 40 or more fragments or pieces are placed in each container forthe first expansion. In some embodiments, the tumor is fragmented and 30or 40 fragments or pieces are placed in each container for the firstexpansion. In some embodiments, the tumor is fragmented and 40 fragmentsor pieces are placed in each container for the first expansion. In someembodiments, the multiple fragments comprise about 4 to about 50fragments, wherein each fragment has a volume of about 27 mm³. In someembodiments, the multiple fragments comprise about 30 to about 60fragments with a total volume of about 1300 mm³ to about 1500 mm³. Insome embodiments, the multiple fragments comprise about 50 fragmentswith a total volume of about 1350 mm³. In some embodiments, the multiplefragments comprise about 50 fragments with a total mass of about 1 gramto about 1.5 grams. In some embodiments, the multiple fragments compriseabout 4 fragments.

In some embodiments, the TILs are obtained from tumor fragments. In someembodiments, the tumor fragment is obtained by sharp dissection. In someembodiments, the tumor fragment is between about 1 mm³ and 10 mm³. Insome embodiments, the tumor fragment is between about 1 mm³ and 8 mm³.In some embodiments, the tumor fragment is about 1 mm³. In someembodiments, the tumor fragment is about 2 mm³. In some embodiments, thetumor fragment is about 3 mm³. In some embodiments, the tumor fragmentis about 4 mm³. In some embodiments, the tumor fragment is about 5 mm³.In some embodiments, the tumor fragment is about 6 mm³. In someembodiments, the tumor fragment is about 7 mm³. In some embodiments, thetumor fragment is about 8 mm³. In some embodiments, the tumor fragmentis about 9 mm³. In some embodiments, the tumor fragment is about 10 mm³.

In some embodiments, the TILs are obtained from tumor digests. In someembodiments, tumor digests were generated by incubation in enzyme media,for example but not limited to RPMI 1640, 2 mM GlutaMAX, 10 mg/mLgentamicin, 30 U/mL DNase, and 1.0 mg/mL collagenase, followed bymechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, Calif.).After placing the tumor in enzyme media, the tumor can be mechanicallydissociated for approximately 1 minute. The solution can then beincubated for 30 minutes at 37° C. in 5% CO₂ and it then mechanicallydisrupted again for approximately 1 minute. After being incubated againfor 30 minutes at 37° C. in 5% CO₂, the tumor can be mechanicallydisrupted a third time for approximately 1 minute. In some embodiments,after the third mechanical disruption if large pieces of tissue werepresent, 1 or 2 additional mechanical dissociations were applied to thesample, with or without 30 additional minutes of incubation at 37° C. in5% CO₂. In some embodiments, at the end of the final incubation if thecell suspension contained a large number of red blood cells or deadcells, a density gradient separation using Ficoll can be performed toremove these cells.

In some embodiments, the harvested cell suspension prior to the firstexpansion step is called a “primary cell population” or a “freshlyharvested” cell population.

In some embodiments, cells can be optionally frozen after sample harvestand stored frozen prior to entry into the expansion described in Step B,which is described in further detail below, as well as exemplified inFIG. 27.

B. Step B: First Expansion

1. Young TILs

In some embodiments, the present methods provide for obtaining youngTILs, which are capable of increased replication cycles uponadministration to a subject/patient and as such may provide additionaltherapeutic benefits over older TILs (i.e., TILs which have furtherundergone more rounds of replication prior to administration to asubject/patient). Features of young TILs have been described in theliterature, for example Donia, at al., Scandinavian Journal ofImmunology, 75:157-167 (2012); Dudley et al., Clin Cancer Res,16:6122-6131 (2010); Huang et al., J Immunother, 28(3):258-267 (2005);Besser et al., Clin Cancer Res, 19(17):OF1-OF9 (2013); Besser et al., JImmunother 32:415-423 (2009); Robbins, et al., J Immunol 2004;173:7125-7130; Shen et al., J Immunother, 30:123-129 (2007); Zhou, etal., J Immunother, 28:53-62 (2005); and Tran, et al., J Immunother,31:742-751 (2008), all of which are incorporated herein by reference intheir entireties.

The diverse antigen receptors of T and B lymphocytes are produced bysomatic recombination of a limited, but large number of gene segments.These gene segments: V (variable), D (diversity), J (joining), and C(constant), determine the binding specificity and downstreamapplications of immunoglobulins and T-cell receptors (TCRs). The presentinvention provides a method for generating TILs which exhibit andincrease the T-cell repertoire diversity. In some embodiments, the TILsobtained by the present method exhibit an increase in the T-cellrepertoire diversity. In some embodiments, the TILs obtained by thepresent method exhibit an increase in the T-cell repertoire diversity ascompared to freshly harvested TILs and/or TILs prepared using othermethods than those provide herein including for example, methods otherthan those embodied in FIG. 27. In some embodiments, the TILs obtainedby the present method exhibit an increase in the T-cell repertoirediversity as compared to freshly harvested TILs and/or TILs preparedusing methods referred to as process 1C, as exemplified in FIG. 83. Insome embodiments, the TILs obtained in the first expansion exhibit anincrease in the T-cell repertoire diversity. In some embodiments, theincrease in diversity is an increase in the immunoglobulin diversityand/or the T-cell receptor diversity. In some embodiments, the diversityis in the immunoglobulin is in the immunoglobulin heavy chain. In someembodiments, the diversity is in the immunoglobulin is in theimmunoglobulin light chain. In some embodiments, the diversity is in theT-cell receptor. In some embodiments, the diversity is in one of theT-cell receptors selected from the group consisting of alpha, beta,gamma, and delta receptors. In some embodiments, there is an increase inthe expression of T-cell receptor (TCR) alpha and/or beta. In someembodiments, there is an increase in the expression of T-cell receptor(TCR) alpha. In some embodiments, there is an increase in the expressionof T-cell receptor (TCR) beta. In some embodiments, there is an increasein the expression of TCRab (i.e., TCRα/β).

After dissection or digestion of tumor fragments, for example such asdescribed in Step A of FIG. 27, the resulting cells are cultured inserum containing IL-2 under conditions that favor the growth of TILsover tumor and other cells. In some embodiments, the tumor digests areincubated in 2 mL wells in media comprising inactivated human AB serumwith 6000 IU/mL of IL-2. This primary cell population is cultured for aperiod of days, generally from 3 to 14 days, resulting in a bulk TILpopulation, generally about 1×10⁸ bulk TIL cells. In some embodiments,this primary cell population is cultured for a period of 7 to 14 days,resulting in a bulk TIL population, generally about 1×10⁸ bulk TILcells. In some embodiments, this primary cell population is cultured fora period of 10 to 14 days, resulting in a bulk TIL population, generallyabout 1×10⁸ bulk TIL cells. In some embodiments, this primary cellpopulation is cultured for a period of about 11 days, resulting in abulk TIL population, generally about 1×10⁸ bulk TIL cells.

In a preferred embodiment, expansion of TILs may be performed using aninitial bulk TIL expansion step (for example such as those described inStep B of FIG. 27, which can include processes referred to as pre-REP)as described below and herein, followed by a second expansion (Step D,including processes referred to as rapid expansion protocol (REP) steps)as described below under Step D and herein, followed by optionalcryopreservation, and followed by a second Step D (including processesreferred to as restimulation REP steps) as described below and herein.The TILs obtained from this process may be optionally characterized forphenotypic characteristics and metabolic parameters as described herein.

In embodiments where TIL cultures are initiated in 24-well plates, forexample, using Costar 24-well cell culture cluster, flat bottom (CorningIncorporated, Corning, N.Y., each well can be seeded with 1×10⁶ tumordigest cells or one tumor fragment in 2 mL of complete medium (CM) withIL-2 (6000 IU/mL; Chiron Corp., Emeryville, Calif.). In someembodiments, the tumor fragment is between about 1 mm³ and 10 mm³.

In some embodiments, the first expansion culture medium is referred toas “CM”, an abbreviation for culture media. In some embodiments, CM forStep B consists of RPMI 1640 with GlutaMAX, supplemented with 10% humanAB serum, 25 mM Hepes, and 10 mg/mL gentamicin. In embodiments wherecultures are initiated in gas-permeable flasks with a 40 mL capacity anda 10 cm² gas-permeable silicon bottom (for example, G-Rex10; Wilson WolfManufacturing, New Brighton, Minn.) (FIG. 1), each flask was loaded with10-40×10⁶ viable tumor digest cells or 5-30 tumor fragments in 10-40 mLof CM with IL-2. Both the G-Rex10 and 24-well plates were incubated in ahumidified incubator at 37° C. in 5% CO₂ and 5 days after cultureinitiation, half the media was removed and replaced with fresh CM andIL-2 and after day 5, half the media was changed every 2-3 days.

After preparation of the tumor fragments, the resulting cells (i.e.,fragments) are cultured in serum containing IL-2 under conditions thatfavor the growth of TILs over tumor and other cells. In someembodiments, the tumor digests are incubated in 2 mL wells in mediacomprising inactivated human AB serum (or, in some cases, as outlinedherein, in the presence of aAPC cell population) with 6000 IU/mL ofIL-2. This primary cell population is cultured for a period of days,generally from 10 to 14 days, resulting in a bulk TIL population,generally about 1×10⁸ bulk TIL cells. In some embodiments, the growthmedia during the first expansion comprises IL-2 or a variant thereof. Insome embodiments, the IL is recombinant human IL-2 (rhIL-2). In someembodiments the IL-2 stock solution has a specific activity of 20-30×10⁶IU/mg for a 1 mg vial. In some embodiments the IL-2 stock solution has aspecific activity of 20×10⁶ IU/mg for a 1 mg vial. In some embodimentsthe IL-2 stock solution has a specific activity of 25×10⁶ IU/mg for a 1mg vial. In some embodiments the IL-2 stock solution has a specificactivity of 30×10⁶ IU/mg for a 1 mg vial. In some embodiments, the IL-2stock solution has a final concentration of 4-8×10⁶ IU/mg of IL-2. Insome embodiments, the IL-2 stock solution has a final concentration of5-7×10⁶ IU/mg of IL-2. In some embodiments, the IL-2 stock solution hasa final concentration of 6×10⁶ IU/mg of IL-2. In some embodiments, theIL-2 stock solution is prepare as described in Example 4. In someembodiments, the first expansion culture media comprises about 10,000IU/mL of IL-2, about 9,000 IU/mL of IL-2, about 8,000 IU/mL of IL-2,about 7,000 IU/mL of IL-2, about 6000 IU/mL of IL-2 or about 5,000 IU/mLof IL-2. In some embodiments, the first expansion culture mediacomprises about 9,000 IU/mL of IL-2 to about 5,000 IU/mL of IL-2. Insome embodiments, the first expansion culture media comprises about8,000 IU/mL of IL-2 to about 6,000 IU/mL of IL-2. In some embodiments,the first expansion culture media comprises about 7,000 IU/mL of IL-2 toabout 6,000 IU/mL of IL-2. In some embodiments, the first expansionculture media comprises about 6,000 IU/mL of IL-2. In an embodiment, thecell culture medium further comprises IL-2. In some embodiments, thecell culture medium comprises about 3000 IU/mL of IL-2. In anembodiment, the cell culture medium further comprises IL-2. In apreferred embodiment, the cell culture medium comprises about 3000 IU/mLof IL-2. In an embodiment, the cell culture medium comprises about 1000IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000IU/mL, about 7500 IU/mL, or about 8000 IU/mL of IL-2. In an embodiment,the cell culture medium comprises between 1000 and 2000 IU/mL, between2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between 4000 and 5000IU/mL, between 5000 and 6000 IU/mL, between 6000 and 7000 IU/mL, between7000 and 8000 IU/mL, or about 8000 IU/mL of IL-2.

In some embodiments, first expansion culture media comprises about 500IU/mL of IL-15, about 400 IU/mL of IL-15, about 300 IU/mL of IL-15,about 200 IU/mL of IL-15, about 180 IU/mL of IL-15, about 160 IU/mL ofIL-15, about 140 IU/mL of IL-15, about 120 IU/mL of IL-15, or about 100IU/mL of IL-15. In some embodiments, the first expansion culture mediacomprises about 500 IU/mL of IL-15 to about 100 IU/mL of IL-15. In someembodiments, the first expansion culture media comprises about 400 IU/mLof IL-15 to about 100 IU/mL of IL-15. In some embodiments, the firstexpansion culture media comprises about 300 IU/mL of IL-15 to about 100IU/mL of IL-15. In some embodiments, the first expansion culture mediacomprises about 200 IU/mL of IL-15. In some embodiments, the cellculture medium comprises about 180 IU/mL of IL-15. In an embodiment, thecell culture medium further comprises IL-15. In a preferred embodiment,the cell culture medium comprises about 180 IU/mL of IL-15.

In some embodiments, first expansion culture media comprises about 20IU/mL of IL-21, about 15 IU/mL of IL-21, about 12 IU/mL of IL-21, about10 IU/mL of IL-21, about 5 IU/mL of IL-21, about 4 IU/mL of IL-21, about3 IU/mL of IL-21, about 2 IU/mL of IL-21, about 1 IU/mL of IL-21, orabout 0.5 IU/mL of IL-21. In some embodiments, the first expansionculture media comprises about 20 IU/mL of IL-21 to about 0.5 IU/mL ofIL-21. In some embodiments, the first expansion culture media comprisesabout 15 IU/mL of IL-21 to about 0.5 IU/mL of IL-21. In someembodiments, the first expansion culture media comprises about 12 IU/mLof IL-21 to about 0.5 IU/mL of IL-21. In some embodiments, the firstexpansion culture media comprises about 10 IU/mL of IL-21 to about 0.5IU/mL of IL-21. In some embodiments, the first expansion culture mediacomprises about 5 IU/mL of IL-21 to about 1 IU/mL of IL-21. In someembodiments, the first expansion culture media comprises about 2 IU/mLof IL-21. In some embodiments, the cell culture medium comprises about 1IU/mL of IL-21. In some embodiments, the cell culture medium comprisesabout 0.5 IU/mL of IL-21. In an embodiment, the cell culture mediumfurther comprises IL-21. In a preferred embodiment, the cell culturemedium comprises about 1 IU/mL of IL-21.

In some embodiments, the first expansion culture medium is referred toas “CM”, an abbreviation for culture media. In some embodiments, it isreferred to as CM1 (culture medium 1). In some embodiments, CM consistsof RPMI 1640 with GlutaMAX, supplemented with 10% human AB serum, 25 mMHepes, and 10 mg/mL gentamicin. In embodiments where cultures areinitiated in gas-permeable flasks with a 40 mL capacity and a 10 cm²gas-permeable silicon bottom (for example, G-Rex10; Wilson WolfManufacturing, New Brighton, Minn.) (FIG. 1), each flask was loaded with10-40×10⁶ viable tumor digest cells or 5-30 tumor fragments in 10-40 mLof CM with IL-2. Both the G-Rex10 and 24-well plates were incubated in ahumidified incubator at 37° C. in 5% CO₂ and 5 days after cultureinitiation, half the media was removed and replaced with fresh CM andIL-2 and after day 5, half the media was changed every 2-3 days. In someembodiments, the CM is the CM1 described in the Examples, see, Example5. In some embodiments, the first expansion occurs in an initial cellculture medium or a first cell culture medium. In some embodiments, theinitial cell culture medium or the first cell culture medium comprisesIL-2.

In some embodiments, the first expansion (including processes such asfor example those described in Step B of FIG. 27, which can includethose sometimes referred to as the pre-REP) process is shortened to 3-14days, as discussed in the examples and figures. In some embodiments, thefirst expansion (including processes such as for example those describedin Step B of FIG. 27, which can include those sometimes referred to asthe pre-REP) is shortened to 7 to 14 days, as discussed in the Examplesand shown in FIGS. 4 and 5, as well as including for example, anexpansion as described in Step B of FIG. 27. In some embodiments, thefirst expansion of Step B is shortened to 10-14 days, as discussed inthe Examples and shown in FIGS. 4 and 5. In some embodiments, the firstexpansion is shortened to 11 days, as discussed in the Examples andshown in FIGS. 4 and 5, as well as including for example, an expansionas described in Step B of FIG. 27.

In some embodiments, the first TIL expansion can proceed for 1 day, 2days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days,11 days, 12 days, 13 days, or 14 days. In some embodiments, the firstTIL expansion can proceed for 1 day to 14 days. In some embodiments, thefirst TIL expansion can proceed for 2 days to 14 days. In someembodiments, the first TIL expansion can proceed for 3 days to 14 days.In some embodiments, the first TIL expansion can proceed for 4 days to14 days. In some embodiments, the first TIL expansion can proceed for 5days to 14 days. In some embodiments, the first TIL expansion canproceed for 6 days to 14 days. In some embodiments, the first TILexpansion can proceed for 7 days to 14 days. In some embodiments, thefirst TIL expansion can proceed for 8 days to 14 days. In someembodiments, the first TIL expansion can proceed for 9 days to 14 days.In some embodiments, the first TIL expansion can proceed for 10 days to14 days. In some embodiments, the first TIL expansion can proceed for 11days to 14 days. In some embodiments, the first TIL expansion canproceed for 12 days to 14 days. In some embodiments, the first TILexpansion can proceed for 13 days to 14 days. In some embodiments, thefirst TIL expansion can proceed for 14 days. In some embodiments, thefirst TIL expansion can proceed for 1 day to 11 days. In someembodiments, the first TIL expansion can proceed for 2 days to 11 days.In some embodiments, the first TIL expansion can proceed for 3 days to11 days. In some embodiments, the first TIL expansion can proceed for 4days to 11 days. In some embodiments, the first TIL expansion canproceed for 5 days to 11 days. In some embodiments, the first TILexpansion can proceed for 6 days to 11 days. In some embodiments, thefirst TIL expansion can proceed for 7 days to 11 days. In someembodiments, the first TIL expansion can proceed for 8 days to 11 days.In some embodiments, the first TIL expansion can proceed for 9 days to11 days. In some embodiments, the first TIL expansion can proceed for 10days to 11 days. In some embodiments, the first TIL expansion canproceed for 11 days.

In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21are employed as a combination during the first expansion. In someembodiments, IL-2, IL-7, IL-15, and/or IL-21 as well as any combinationsthereof can be included during the first expansion, including forexample during a Step B processes according to FIG. 27, as well asdescribed herein. In some embodiments, a combination of IL-2, IL-15, andIL-21 are employed as a combination during the first expansion. In someembodiments, IL-2, IL-15, and IL-21 as well as any combinations thereofcan be included during Step B processes according to FIG. 27 and asdescribed herein.

In some embodiments, the first expansion (including processes referredto as the pre-REP; for example, Step B according to FIG. 27) process isshortened to 3 to 14 days, as discussed in the examples and figures. Insome embodiments, the first expansion of Step B is shortened to 7 to 14days, as discussed in the Examples and shown in FIGS. 4 and 5. In someembodiments, the first expansion of Step B is shortened to 10 to 14days, as discussed in the Examples and shown in FIGS. 4, 5, and 27. Insome embodiments, the first expansion is shortened to 11 days, asdiscussed in the Examples and shown in FIGS. 4, 5, and 27.

In some embodiments, the first expansion, for example, Step B accordingto FIG. 27, is performed in a closed system bioreactor. In someembodiments, a closed system is employed for the TIL expansion, asdescribed herein. In some embodiments, a single bioreactor is employed.In some embodiments, the single bioreactor employed is for example aG-REX-10 or a G-REX-100. In some embodiments, the closed systembioreactor is a single bioreactor.

C. Step C: First Expansion to Second Expansion Transition

In some cases, the bulk TIL population obtained from the firstexpansion, including for example the TIL population obtained from forexample, Step B as indicated in FIG. 27, can be cryopreservedimmediately, using the protocols discussed herein below. Alternatively,the TIL population obtained from the first expansion, referred to as thesecond TIL population, can be subjected to a second expansion (which caninclude expansions sometimes referred to as REP) and then cryopreservedas discussed below. Similarly, in the case where genetically modifiedTILs will be used in therapy, the first TIL population (sometimesreferred to as the bulk TIL population) or the second TIL population(which can in some embodiments include populations referred to as theREP TIL populations) can be subjected to genetic modifications forsuitable treatments prior to expansion or after the first expansion andprior to the second expansion.

In some embodiments, the TILs obtained from the first expansion (forexample, from Step B as indicated in FIG. 27) are stored untilphenotyped for selection. In some embodiments, the TILs obtained fromthe first expansion (for example, from Step B as indicated in FIG. 27)are not stored and proceed directly to the second expansion. In someembodiments, the TILs obtained from the first expansion are notcryopreserved after the first expansion and prior to the secondexpansion. In some embodiments, the transition from the first expansionto the second expansion occurs at about 3 days, 4, days, 5 days, 6 days,7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs at about 3 days to 14days from when fragmentation occurs. In some embodiments, the transitionfrom the first expansion to the second expansion occurs at about 4 daysto 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs atabout 4 days to 10 days from when fragmentation occurs. In someembodiments, the transition from the first expansion to the secondexpansion occurs at about 7 days to 14 days from when fragmentationoccurs. In some embodiments, the transition from the first expansion tothe second expansion occurs at about 14 days from when fragmentationoccurs.

In some embodiments, the transition from the first expansion to thesecond expansion occurs at 1 day, 2 days, 3 days, 4 days, 5 days, 6days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14days from when fragmentation occurs. In some embodiments, the transitionfrom the first expansion to the second expansion occurs 1 day to 14 daysfrom when fragmentation occurs. In some embodiments, the first TILexpansion can proceed for 2 days to 14 days. In some embodiments, thetransition from the first expansion to the second expansion occurs 3days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 4days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 5days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 6days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 7days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 8days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 9days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 10days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 11days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 12days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 13days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 14days from when fragmentation occurs. In some embodiments, the transitionfrom the first expansion to the second expansion occurs 1 day to 11 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs 2 days to 11 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs 3 days to 11 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs 4 days to 11 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs 5 days to 11 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs 6 days to 11 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs 7 days to 11 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs 8 days to 11 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs 9 days to 11 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs 10 days to 11 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs 11 days from whenfragmentation occurs.

In some embodiments, the TILs are not stored after the first expansionand prior to the second expansion, and the TILs proceed directly to thesecond expansion (for example, in some embodiments, there is no storageduring the transition from Step B to Step D as shown in FIG. 27). Insome embodiments, the transition occurs in closed system, as describedherein. In some embodiments, the TILs from the first expansion, thesecond population of TILs, proceeds directly into the second expansionwith no transition period.

In some embodiments, the transition from the first expansion to thesecond expansion, for example, Step C according to FIG. 27, is performedin a closed system bioreactor. In some embodiments, a closed system isemployed for the TIL expansion, as described herein. In someembodiments, a single bioreactor is employed. In some embodiments, thesingle bioreactor employed is for example a G-REX-10 or a G-REX-100. Insome embodiments, the closed system bioreactor is a single bioreactor.

D. Step D: Second Expansion

In some embodiments, the TIL cell population is expanded in number afterharvest and initial bulk processing for example, after Step A and StepB, and the transition referred to as Step C, as indicated in FIG. 27).This further expansion is referred to herein as the second expansion,which can include expansion processes generally referred to in the artas a rapid expansion process (REP; as well as processes as indicated inStep D of FIG. 27). The second expansion is generally accomplished usinga culture media comprising a number of components, including feedercells, a cytokine source, and an anti-CD3 antibody, in a gas-permeablecontainer.

In some embodiments, the second expansion or second TIL expansion (whichcan include expansions sometimes referred to as REP; as well asprocesses as indicated in Step D of FIG. 27) of TIL can be performedusing any TIL flasks or containers known by those of skill in the art.In some embodiments, the second TIL expansion can proceed for 7 days, 8days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In someembodiments, the second TIL expansion can proceed for about 7 days toabout 14 days. In some embodiments, the second TIL expansion can proceedfor about 8 days to about 14 days. In some embodiments, the second TILexpansion can proceed for about 9 days to about 14 days. In someembodiments, the second TIL expansion can proceed for about 10 days toabout 14 days. In some embodiments, the second TIL expansion can proceedfor about 11 days to about 14 days. In some embodiments, the second TILexpansion can proceed for about 12 days to about 14 days. In someembodiments, the second TIL expansion can proceed for about 13 days toabout 14 days. In some embodiments, the second TIL expansion can proceedfor about 14 days.

In an embodiment, the second expansion can be performed in a gaspermeable container using the methods of the present disclosure(including for example, expansions referred to as REP; as well asprocesses as indicated in Step D of FIG. 27). For example, TILs can berapidly expanded using non-specific T-cell receptor stimulation in thepresence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Thenon-specific T-cell receptor stimulus can include, for example, ananti-CD3 antibody, such as about 30 ng/ml of OKT3, a mouse monoclonalanti-CD3 antibody (commercially available from Ortho-McNeil, Raritan,N.J. or Miltenyi Biotech, Auburn, Calif.) or UHCT-1 (commerciallyavailable from BioLegend, San Diego, Calif., USA). TILs can be expandedto induce further stimulation of the TILs in vitro by including one ormore antigens during the second expansion, including antigenic portionsthereof, such as epitope(s), of the cancer, which can be optionallyexpressed from a vector, such as a human leukocyte antigen A2 (HLA-A2)binding peptide, e.g., 0.3 μM MART-1:26-35 (27 L) or gpl 00:209-217(210M), optionally in the presence of a T-cell growth factor, such as300 IU/mL IL-2 or IL-15. Other suitable antigens may include, e.g.,NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, andVEGFR2, or antigenic portions thereof. TIL may also be rapidly expandedby re-stimulation with the same antigen(s) of the cancer pulsed ontoHLA-A2-expressing antigen-presenting cells. Alternatively, the TILs canbe further re-stimulated with, e.g., example, irradiated, autologouslymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.In some embodiments, the re-stimulation occurs as part of the secondexpansion. In some embodiments, the second expansion occurs in thepresence of irradiated, autologous lymphocytes or with irradiatedHLA-A2+ allogeneic lymphocytes and IL-2.

In an embodiment, the cell culture medium further comprises IL-2. In asome embodiments, the cell culture medium comprises about 3000 IU/mL ofIL-2. In an embodiment, the cell culture medium comprises about 1000IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000IU/mL, about 7500 IU/mL, or about 8000 IU/mL of IL-2. In an embodiment,the cell culture medium comprises between 1000 and 2000 IU/mL, between2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between 4000 and 5000IU/mL, between 5000 and 6000 IU/mL, between 6000 and 7000 IU/mL, between7000 and 8000 IU/mL, or between 8000 IU/mL of IL-2.

In an embodiment, the cell culture medium comprises OKT3 antibody. In asome embodiments, the cell culture medium comprises about 30 ng/mL ofOKT3 antibody. In an embodiment, the cell culture medium comprises about0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5ng/mL, about 7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL,about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL,about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, and about 1 μg/mL ofOKT3 antibody. In an embodiment, the cell culture medium comprisesbetween 0.1 ng/mL and 1 ng/mL, between 1 ng/mL and 5 ng/mL, between 5ng/mL and 10 ng/mL, between 10 ng/mL and 20 ng/mL, between 20 ng/mL and30 ng/mL, between 30 ng/mL and 40 ng/mL, between 40 ng/mL and 50 ng/mL,and between 50 ng/mL and 100 ng/mL of OKT3 antibody.

In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21are employed as a combination during the second expansion. In someembodiments, IL-2, IL-7, IL-15, and/or IL-21 as well as any combinationsthereof can be included during the second expansion, including forexample during a Step D processes according to FIG. 27, as well asdescribed herein. In some embodiments, a combination of IL-2, IL-15, andIL-21 are employed as a combination during the second expansion. In someembodiments, IL-2, IL-15, and IL-21 as well as any combinations thereofcan be included during Step D processes according to FIG. 27 and asdescribed herein.

In some embodiments, the second expansion can be conducted in asupplemented cell culture medium comprising IL-2, OKT-3, andantigen-presenting feeder cells. In some embodiments, the secondexpansion occurs in a supplemented cell culture medium. In someembodiments, the supplemented cell culture medium comprises IL-2, OKT-3,and antigen-presenting feeder cells. In some embodiments, the secondcell culture medium comprises IL-2, OKT-3, and antigen-presenting cells(APCs; also referred to as antigen-presenting feeder cells). In someembodiments, the second expansion occurs in a cell culture mediumcomprising IL-2, OKT-3, and antigen-presenting feeder cells (i.e.,antigen presenting cells).

In some embodiments, the second expansion culture media comprises about500 IU/mL of IL-15, about 400 IU/mL of IL-15, about 300 IU/mL of IL-15,about 200 IU/mL of IL-15, about 180 IU/mL of IL-15, about 160 IU/mL ofIL-15, about 140 IU/mL of IL-15, about 120 IU/mL of IL-15, or about 100IU/mL of IL-15. In some embodiments, the second expansion culture mediacomprises about 500 IU/mL of IL-15 to about 100 IU/mL of IL-15. In someembodiments, the second expansion culture media comprises about 400IU/mL of IL-15 to about 100 IU/mL of IL-15. In some embodiments, thesecond expansion culture media comprises about 300 IU/mL of IL-15 toabout 100 IU/mL of IL-15. In some embodiments, the second expansionculture media comprises about 200 IU/mL of IL-15. In some embodiments,the cell culture medium comprises about 180 IU/mL of IL-15. In anembodiment, the cell culture medium further comprises IL-15. In apreferred embodiment, the cell culture medium comprises about 180 IU/mLof IL-15.

In some embodiments, the second expansion culture media comprises about20 IU/mL of IL-21, about 15 IU/mL of IL-21, about 12 IU/mL of IL-21,about 10 IU/mL of IL-21, about 5 IU/mL of IL-21, about 4 IU/mL of IL-21,about 3 IU/mL of IL-21, about 2 IU/mL of IL-21, about 1 IU/mL of IL-21,or about 0.5 IU/mL of IL-21. In some embodiments, the second expansionculture media comprises about 20 IU/mL of IL-21 to about 0.5 IU/mL ofIL-21. In some embodiments, the second expansion culture media comprisesabout 15 IU/mL of IL-21 to about 0.5 IU/mL of IL-21. In someembodiments, the second expansion culture media comprises about 12 IU/mLof IL-21 to about 0.5 IU/mL of IL-21. In some embodiments, the secondexpansion culture media comprises about 10 IU/mL of IL-21 to about 0.5IU/mL of IL-21. In some embodiments, the second expansion culture mediacomprises about 5 IU/mL of IL-21 to about 1 IU/mL of IL-21. In someembodiments, the second expansion culture media comprises about 2 IU/mLof IL-21. In some embodiments, the cell culture medium comprises about 1IU/mL of IL-21. In some embodiments, the cell culture medium comprisesabout 0.5 IU/mL of IL-21. In an embodiment, the cell culture mediumfurther comprises IL-21. In a preferred embodiment, the cell culturemedium comprises about 1 IU/mL of IL-21.

In some embodiments the antigen-presenting feeder cells (APCs) arePBMCs. In an embodiment, the ratio of TILs to PBMCs and/orantigen-presenting cells in the rapid expansion and/or the secondexpansion is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225,about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1to 350, about 1 to 375, about 1 to 400, or about 1 to 500. In anembodiment, the ratio of TILs to PBMCs in the rapid expansion and/or thesecond expansion is between 1 to 50 and 1 to 300. In an embodiment, theratio of TILs to PBMCs in the rapid expansion and/or the secondexpansion is between 1 to 100 and 1 to 200.

In an embodiment, REP and/or the second expansion is performed in flaskswith the bulk TILs being mixed with a 100- or 200-fold excess ofinactivated feeder cells, 30 mg/mL OKT3 anti-CD3 antibody and 3000 IU/mLIL-2 in 150 ml media. Media replacement is done (generally 2/3 mediareplacement via respiration with fresh media) until the cells aretransferred to an alternative growth chamber. Alternative growthchambers include G-REX flasks and gas permeable containers as more fullydiscussed below.

In some embodiments, the second expansion (which can include processesreferred to as the REP process) is shortened to 7-14 days, as discussedin the examples and figures. In some embodiments, the second expansionis shortened to 11 days.

In an embodiment, REP and/or the second expansion may be performed usingT-175 flasks and gas permeable bags as previously described (Tran, etal., J. Immunother. 2008, 31, 742-51; Dudley, et al., J. Immunother.2003, 26, 332-42) or gas permeable cultureware (G-Rex flasks). In someembodiments, the second expansion (including expansions referred to asrapid expansions) is performed in T-175 flasks, and about 1×10⁶ TILssuspended in 150 mL of media may be added to each T-175 flask. The TILsmay be cultured in a 1 to 1 mixture of CM and AIM-V medium, supplementedwith 3000 IU per mL of IL-2 and 30 ng per ml of anti-CD3. The T-175flasks may be incubated at 37° C. in 5% CO₂. Half the media may beexchanged on day 5 using 50/50 medium with 3000 IU per mL of IL-2. Insome embodiments, on day 7 cells from two T-175 flasks may be combinedin a 3 L bag and 300 mL of AIM V with 5% human AB serum and 3000 IU permL of IL-2 was added to the 300 ml of TIL suspension. The number ofcells in each bag was counted every day or two and fresh media was addedto keep the cell count between 0.5 and 2.0×10⁶ cells/mL.

In an embodiment, the second expansion (which can include expansionsreferred to as REP, as well as those referred to in Step D of FIG. 27)may be performed in 500 mL capacity gas permeable flasks with 100 cmgas-permeable silicon bottoms (G-Rex 100, commercially available fromWilson Wolf Manufacturing Corporation, New Brighton, Minn., USA), 5×10⁶or 10×10⁶ TIL may be cultured with PBMCs in 400 mL of 50/50 medium,supplemented with 5% human AB serum, 3000 IU per mL of IL-2 and 30 ngper ml of anti-CD3 (OKT3). The G-Rex 100 flasks may be incubated at 37°C. in 5% CO₂. On day 5, 250 mL of supernatant may be removed and placedinto centrifuge bottles and centrifuged at 1500 rpm (491×g) for 10minutes. The TIL pellets may be re-suspended with 150 mL of fresh mediumwith 5% human AB serum, 3000 IU per mL of IL-2, and added back to theoriginal G-Rex 100 flasks. When TIL are expanded serially in G-Rex 100flasks, on day 7 the TIL in each G-Rex 100 may be suspended in the 300mL of media present in each flask and the cell suspension may be dividedinto 3 100 mL aliquots that may be used to seed 3 G-Rex 100 flasks. Then150 mL of AIM-V with 5% human AB serum and 3000 IU per mL of IL-2 may beadded to each flask. The G-Rex 100 flasks may be incubated at 37° C. in5% CO₂ and after 4 days 150 mL of AIM-V with 3000 IU per mL of IL-2 maybe added to each G-REX 100 flask. The cells may be harvested on day 14of culture.

In an embodiment, the second expansion (including expansions referred toas REP) is performed in flasks with the bulk TILs being mixed with a100- or 200-fold excess of inactivated feeder cells, 30 mg/mL OKT3anti-CD3 antibody and 3000 IU/mL IL-2 in 150 ml media. In someembodiments, media replacement is done until the cells are transferredto an alternative growth chamber. In some embodiments, ⅔ of the media isreplaced by respiration with fresh media. In some embodiments,alternative growth chambers include G-REX flasks and gas permeablecontainers as more fully discussed below.

In an embodiment, the second expansion (including expansions referred toas REP) is performed and further comprises a step wherein TILs areselected for superior tumor reactivity. Any selection method known inthe art may be used. For example, the methods described in U.S. PatentApplication Publication No. 2016/0010058 A1, the disclosures of whichare incorporated herein by reference, may be used for selection of TILsfor superior tumor reactivity.

Optionally, a cell viability assay can be performed after the secondexpansion (including expansions referred to as the REP expansion), usingstandard assays known in the art. For example, a trypan blue exclusionassay can be done on a sample of the bulk TILs, which selectively labelsdead cells and allows a viability assessment. In some embodiments, TILsamples can be counted and viability determined using a Cellometer K2automated cell counter (Nexcelom Bioscience, Lawrence, Mass.). In someembodiments, viability is determined according to the Cellometer K2Image Cytometer Automatic Cell Counter protocol described, for example,in Example 15.

In some embodiments, the second expansion (including expansions referredto as REP) of TIL can be performed using T-175 flasks and gas-permeablebags as previously described (Tran K Q, Zhou J, Durflinger K H, et al.,2008, J Immunother., 31:742-751, and Dudley M E, Wunderlich J R, SheltonT E, et al. 2003, J Immunother., 26:332-342) or gas-permeable G-Rexflasks. In some embodiments, the second expansion is performed usingflasks. In some embodiments, the second expansion is performed usinggas-permeable G-Rex flasks. In some embodiments, the second expansion isperformed in T-175 flasks, and about 1×10⁶ TIL are suspended in about150 mL of media and this is added to each T-175 flask. The TIL arecultured with irradiated (50 Gy) allogeneic PBMC as “feeder” cells at aratio of 1 to 100 and the cells were cultured in a 1 to 1 mixture of CMand AIM-V medium (50/50 medium), supplemented with 3000 IU/mL of IL-2and 30 ng/mL of anti-CD3. The T-175 flasks are incubated at 37° C. in 5%CO₂. In some embodiments, half the media is changed on day 5 using 50/50medium with 3000 IU/mL of IL-2. In some embodiments, on day 7, cellsfrom 2 T-175 flasks are combined in a 3 L bag and 300 mL of AIM-V with5% human AB serum and 3000 IU/mL of IL-2 is added to the 300 mL of TILsuspension. The number of cells in each bag can be counted every day ortwo and fresh media can be added to keep the cell count between about0.5 and about 2.0×10⁶ cells/mL.

In some embodiments, the second expansion (including expansions referredto as REP) are performed in 500 mL capacity flasks with 100 cm²gas-permeable silicon bottoms (G-Rex 100, Wilson Wolf) (FIG. 1), about5×10⁶ or 10×10⁶ TIL are cultured with irradiated allogeneic PBMC at aratio of 1 to 100 in 400 mL of 50/50 medium, supplemented with 3000IU/mL of IL-2 and 30 ng/mL of anti-CD3. The G-Rex 100 flasks areincubated at 37° C. in 5% CO₂. In some embodiments, on day 5, 250 mL ofsupernatant is removed and placed into centrifuge bottles andcentrifuged at 1500 rpm (491 g) for 10 minutes. The TIL pellets can thenbe resuspended with 150 mL of fresh 50/50 medium with 3000 IU/mL of IL-2and added back to the original G-Rex 100 flasks. In embodiments whereTILs are expanded serially in G-Rex 100 flasks, on day 7 the TIL in eachG-Rex 100 are suspended in the 300 mL of media present in each flask andthe cell suspension was divided into three 100 mL aliquots that are usedto seed 3 G-Rex 100 flasks. Then 150 mL of AIM-V with 5% human AB serumand 3000 IU/mL of IL-2 is added to each flask. The G-Rex 100 flasks areincubated at 37° C. in 5% CO₂ and after 4 days 150 mL of AIM-V with 3000IU/mL of IL-2 is added to each G-Rex 100 flask. The cells are harvestedon day 14 of culture.

The diverse antigen receptors of T and B lymphocytes are produced bysomatic recombination of a limited, but large number of gene segments.These gene segments: V (variable), D (diversity), J (joining), and C(constant), determine the binding specificity and downstreamapplications of immunoglobulins and T-cell receptors (TCRs). The presentinvention provides a method for generating TILs which exhibit andincrease the T-cell repertoire diversity. In some embodiments, the TILsobtained by the present method exhibit an increase in the T-cellrepertoire diversity. In some embodiments, the TILs obtained in thesecond expansion exhibit an increase in the T-cell repertoire diversity.In some embodiments, the increase in diversity is an increase in theimmunoglobulin diversity and/or the T-cell receptor diversity. In someembodiments, the diversity is in the immunoglobulin is in theimmunoglobulin heavy chain. In some embodiments, the diversity is in theimmunoglobulin is in the immunoglobulin light chain. In someembodiments, the diversity is in the T-cell receptor. In someembodiments, the diversity is in one of the T-cell receptors selectedfrom the group consisting of alpha, beta, gamma, and delta receptors. Insome embodiments, there is an increase in the expression of T-cellreceptor (TCR) alpha and/or beta. In some embodiments, there is anincrease in the expression of T-cell receptor (TCR) alpha. In someembodiments, there is an increase in the expression of T-cell receptor(TCR) beta. In some embodiments, there is an increase in the expressionof TCRab (i.e., TCRα/β).

In some embodiments, the second expansion culture medium (e.g.,sometimes referred to as CM2 or the second cell culture medium),comprises IL-2, OKT-3, as well as the antigen-presenting feeder cells(APCs), as discussed in more detail below.

In some embodiments, the second expansion, for example, Step D accordingto FIG. 27, is performed in a closed system bioreactor. In someembodiments, a closed system is employed for the TIL expansion, asdescribed herein. In some embodiments, a single bioreactor is employed.In some embodiments, the single bioreactor employed is for example aG-REX-10 or a G-REX-100. In some embodiments, the closed systembioreactor is a single bioreactor.

1. Feeder Cells and Antigen Presenting Cells

In an embodiment, the second expansion procedures described herein (forexample including expansion such as those described in Step D from FIG.27, as well as those referred to as REP) require an excess of feedercells during REP TIL expansion and/or during the second expansion. Inmany embodiments, the feeder cells are peripheral blood mononuclearcells (PBMCs) obtained from standard whole blood units from healthyblood donors. The PBMCs are obtained using standard methods such asFicoll-Paque gradient separation.

In general, the allogenic PBMCs are inactivated, either via irradiationor heat treatment, and used in the REP procedures, as described in theexamples, in particular example 14, which provides an exemplary protocolfor evaluating the replication incompetence of irradiate allogeneicPBMCs.

In some embodiments, PBMCs are considered replication incompetent andaccepted for use in the TIL expansion procedures described herein if thetotal number of viable cells on day 14 is less than the initial viablecell number put into culture on day 0 of the REP and/or day 0 of thesecond expansion (i.e., the start day of the second expansion). See, forexample, Example 14.

In some embodiments, PBMCs are considered replication incompetent andaccepted for use in the TIL expansion procedures described herein if thetotal number of viable cells, cultured in the presence of OKT3 and IL-2,on day 7 and day 14 has not increased from the initial viable cellnumber put into culture on day 0 of the REP and/or day 0 of the secondexpansion (i.e., the start day of the second expansion). In someembodiments, the PBMCs are cultured in the presence of 30 ng/ml OKT3antibody and 3000 IU/ml IL-2. See, for example, Example 13.

In some embodiments, PBMCs are considered replication incompetent andaccepted for use in the TIL expansion procedures described herein if thetotal number of viable cells, cultured in the presence of OKT3 and IL-2,on day 7 and day 14 has not increased from the initial viable cellnumber put into culture on day 0 of the REP and/or day 0 of the secondexpansion (i.e., the start day of the second expansion). In someembodiments, the PBMCs are cultured in the presence of 5-60 ng/ml OKT3antibody and 1000-6000 IU/ml IL-2. In some embodiments, the PBMCs arecultured in the presence of 10-50 ng/ml OKT3 antibody and 2000-5000IU/ml IL-2. In some embodiments, the PBMCs are cultured in the presenceof 20-40 ng/ml OKT3 antibody and 2000-4000 IU/ml IL-2. In someembodiments, the PBMCs are cultured in the presence of 25-35 ng/ml OKT3antibody and 2500-3500 IU/ml IL-2.

In some embodiments, the antigen-presenting feeder cells are PBMCs. Insome embodiments, the antigen-presenting feeder cells are artificialantigen-presenting feeder cells. In an embodiment, the ratio of TILs toantigen-presenting feeder cells in the second expansion is about 1 to25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375,about 1 to 400, or about 1 to 500. In an embodiment, the ratio of TILsto antigen-presenting feeder cells in the second expansion is between 1to 50 and 1 to 300. In an embodiment, the ratio of TILs toantigen-presenting feeder cells in the second expansion is between 1 to100 and 1 to 200.

In an embodiment, the second expansion procedures described hereinrequire a ratio of about 2.5×10⁹ feeder cells to about 100×10⁶ TILs. Inanother embodiment, the second expansion procedures described hereinrequire a ratio of about 2.5×10⁹ feeder cells to about 50×10⁶ TILs. Inyet another embodiment, the second expansion procedures described hereinrequire about 2.5×10⁹ feeder cells to about 25×10⁶ TILs.

In an embodiment, the second expansion procedures described hereinrequire an excess of feeder cells during the second expansion. In manyembodiments, the feeder cells are peripheral blood mononuclear cells(PBMCs) obtained from standard whole blood units from healthy blooddonors. The PBMCs are obtained using standard methods such asFicoll-Paque gradient separation. In an embodiment, artificialantigen-presenting (aAPC) cells are used in place of PBMCs.

In general, the allogenic PBMCs are inactivated, either via irradiationor heat treatment, and used in the TIL expansion procedures describedherein, including the exemplary procedures described in FIGS. 4, 5, and27.

In an embodiment, artificial antigen presenting cells are used in thesecond expansion as a replacement for, or in combination with, PBMCs.

2. Cytokines

The expansion methods described herein generally use culture media withhigh doses of a cytokine, in particular IL-2, as is known in the art.

Alternatively, using combinations of cytokines for the rapid expansionand or second expansion of TILS is additionally possible, withcombinations of two or more of IL-2, IL-15 and IL-21 as is generallyoutlined in International Publication No. WO 2015/189356 and WInternational Publication No. WO 2015/189357, hereby expresslyincorporated by reference in their entirety. Thus, possible combinationsinclude IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21 and IL-2, IL-15and IL-21, with the latter finding particular use in many embodiments.The use of combinations of cytokines specifically favors the generationof lymphocytes, and in particular T-cells as described therein.

3. Anti-CD3 Antibodies

In some embodiments, the culture media used in expansion methodsdescribed herein (including those referred to as REP, see for example,FIG. 27) also includes an anti-CD3 antibody. An anti-CD3 antibody incombination with IL-2 induces T cell activation and cell division in theTIL population. This effect can be seen with full length antibodies aswell as Fab and F(ab′)2 fragments, with the former being generallypreferred; see, e.g., Tsoukas et al., J. Immunol. 1985, 135, 1719,hereby incorporated by reference in its entirety.

As will be appreciated by those in the art, there are a number ofsuitable anti-human CD3 antibodies that find use in the invention,including anti-human CD3 polyclonal and monoclonal antibodies fromvarious mammals, including, but not limited to, murine, human, primate,rat, and canine antibodies. In particular embodiments, the OKT3 anti-CD3antibody is used (commercially available from Ortho-McNeil, Raritan,N.J. or Miltenyi Biotech, Auburn, Calif.).

E. Step E: Harvest TILS

After the second expansion step, cells can be harvested. In someembodiments the TILs are harvested after one, two, three, four or moreexpansion steps, for example as provided in FIG. 27. In some embodimentsthe TILs are harvested after two expansion steps, for example asprovided in FIG. 27.

TILs can be harvested in any appropriate and sterile manner, includingfor example by centrifugation. Methods for TIL harvesting are well knownin the art and any such know methods can be employed with the presentprocess. In some embodiments, TILS are harvest using an automatedsystem.

Cell harvesters and/or cell processing systems are commerciallyavailable from a variety of sources, including, for example, FreseniusKabi, Tomtec Life Science, Perkin Elmer, and Inotech BiosystemsInternational, Inc. Any cell based harvester can be employed with thepresent methods. In some embodiments, the cell harvester and/or cellprocessing systems is a membrane-based cell harvester. In someembodiments, cell harvesting is via a cell processing system, such asthe LOVO system (manufactured by Fresenius Kabi). The term “LOVO cellprocessing system” also refers to any instrument or device manufacturedby any vendor that can pump a solution comprising cells through amembrane or filter such as a spinning membrane or spinning filter in asterile and/or closed system environment, allowing for continuous flowand cell processing to remove supernatant or cell culture media withoutpelletization. In some embodiments, the cell harvester and/or cellprocessing system can perform cell separation, washing, fluid-exchange,concentration, and/or other cell processing steps in a closed, sterilesystem.

In some embodiments, the harvest, for example, Step E according to FIG.27, is performed from a closed system bioreactor. In some embodiments, aclosed system is employed for the TIL expansion, as described herein. Insome embodiments, a single bioreactor is employed. In some embodiments,the single bioreactor employed is for example a G-REX-10 or a G-REX-100.In some embodiments, the closed system bioreactor is a singlebioreactor.

F. Step F: Final Formulation/Transfer to Infusion Bag

After Steps A through E as provided in an exemplary order in FIG. 27 andas outlined in detailed above and herein are complete, cells aretransferred to a container for use in administration to a patient. Insome embodiments, once a therapeutically sufficient number of TILs areobtained using the expansion methods described above, they aretransferred to a container for use in administration to a patient.

In an embodiment, TILs expanded using APCs of the present disclosure areadministered to a patient as a pharmaceutical composition. In anembodiment, the pharmaceutical composition is a suspension of TILs in asterile buffer. TILs expanded using PBMCs of the present disclosure maybe administered by any suitable route as known in the art. In someembodiments, the T-cells are administered as a single intra-arterial orintravenous infusion, which preferably lasts approximately 30 to 60minutes. Other suitable routes of administration includeintraperitoneal, intrathecal, and intralymphatic.

1. Pharmaceutical Compositions, Dosages, and Dosing Regimens

In an embodiment, TILs expanded using the methods of the presentdisclosure are administered to a patient as a pharmaceuticalcomposition. In an embodiment, the pharmaceutical composition is asuspension of TILs in a sterile buffer. TILs expanded using PBMCs of thepresent disclosure may be administered by any suitable route as known inthe art. In some embodiments, the T-cells are administered as a singleintra-arterial or intravenous infusion, which preferably lastsapproximately 30 to 60 minutes. Other suitable routes of administrationinclude intraperitoneal, intrathecal, and intralymphatic administration.

Any suitable dose of TILs can be administered. In some embodiments, fromabout 2.3×10¹⁰ to about 13.7×10¹⁰ TILs are administered, with an averageof around 7.8×10¹⁰ TILs, particularly if the cancer is melanoma. In anembodiment, about 1.2×10¹⁰ to about 4.3×10¹⁰ of TILs are administered.In some embodiments, about 3×10¹⁰ to about 12×10¹⁰ TILs areadministered. In some embodiments, about 4×10¹⁰ to about 10×10¹⁰ TILsare administered. In some embodiments, about 5×10¹⁰ to about 8×10¹⁰ TILsare administered. In some embodiments, about 6×10¹⁰ to about 8×10¹⁰ TILsare administered. In some embodiments, about 7×10¹⁰ to about 8×10¹⁰ TILsare administered. In some embodiments, the therapeutically effectivedosage is about 2.3×10¹⁰ to about 13.7×10¹⁰. In some embodiments, thetherapeutically effective dosage is about 7.8×10¹⁰ TILs, particularly ofthe cancer is melanoma. In some embodiments, the therapeuticallyeffective dosage is about 1.2×10¹⁰ to about 4.3×10¹⁰ of TILs. In someembodiments, the therapeutically effective dosage is about 3×10¹⁰ toabout 12×10¹⁰ TILs. In some embodiments, the therapeutically effectivedosage is about 4×10¹⁰ to about 10×10¹⁰ TILs. In some embodiments, thetherapeutically effective dosage is about 5×10¹⁰ to about 8×10¹⁰ TILs.In some embodiments, the therapeutically effective dosage is about6×10¹⁰ to about 8×10¹⁰ TILs. In some embodiments, the therapeuticallyeffective dosage is about 7×10¹⁰ to about 8×10¹⁰ TILs.

In some embodiments, the number of the TILs provided in thepharmaceutical compositions of the invention is about 1×10⁶, 2×10⁶,3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷,4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸,5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹,6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰ 2×10¹⁰ 2×10¹⁰, 3×10¹⁰, 4×10¹⁰,5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹,5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, 1×10¹², 2×10¹², 3×10¹², 4×10¹²,5×10¹², 6×10¹², 7×10¹², 8×10¹², 9×10¹², 1×10¹³, 2×10¹³, 3×10¹³, 4×10¹³,5×10¹³, 6×10¹³, 7×10¹³, 8×10¹³, and 9×10¹³. In an embodiment, the numberof the TILs provided in the pharmaceutical compositions of the inventionis in the range of 1×10⁶ to 5×10⁶, 5×10⁶ to 1×10⁷, 1×10⁷ to 5×10⁷, 5×10⁷to 1×10⁸, 1×10⁸ to 5×10⁸, 5×10⁸ to 1×10⁹, 1×10⁹ to 5×10⁹, 5×10⁹ to1×10¹⁰, 1×10¹⁰ to 5×10¹⁰, 5×10¹⁰ to 1×10¹¹, 5×10¹¹ to 1×10¹², 1×10¹² to5×10¹², and 5×10¹² to 1×10¹³.

In some embodiments, the concentration of the TILs provided in thepharmaceutical compositions of the invention is less than, for example,100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%,14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%,0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%,0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%,0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%,0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v of the pharmaceuticalcomposition.

In some embodiments, the concentration of the TILs provided in thepharmaceutical compositions of the invention is greater than 90%, 80%,70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%,18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25%16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%,13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25%11%, 10.75%, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%,8.25% 8%, 7.75%, 7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%,5.25% 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%,2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%,0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%,0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%,0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001%w/w, w/v, or v/v of the pharmaceutical composition.

In some embodiments, the concentration of the TILs provided in thepharmaceutical compositions of the invention is in the range from about0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about30%, about 0.02% to about 29%, about 0.03% to about 28%, about 0.04% toabout 27%, about 0.05% to about 26%, about 0.06% to about 25%, about0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%,about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% toabout 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9%to about 12% or about 1% to about 10% w/w, w/v or v/v of thepharmaceutical composition.

In some embodiments, the concentration of the TILs provided in thepharmaceutical compositions of the invention is in the range from about0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%,about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% toabout 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w/w, w/vor v/v of the pharmaceutical composition.

In some embodiments, the amount of the TILs provided in thepharmaceutical compositions of the invention is equal to or less than 10g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g,4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g,0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g,0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g,0.0008 g, 0.0007 g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g, or0.0001 g.

In some embodiments, the amount of the TILs provided in thepharmaceutical compositions of the invention is more than 0.0001 g,0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g,0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g,0.008 g, 0.0085 g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g,0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07g, 0.075 g, 0.08 g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25g, 0.3 g, 0.35 g, 0.4 g, 0.45 g, 0.5 g, 0.55 g, 0.6 g, 0.65 g, 0.7 g,0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g, 2.5, 3 g, 3.5, 4g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g, or10 g.

The TILs provided in the pharmaceutical compositions of the inventionare effective over a wide dosage range. The exact dosage will dependupon the route of administration, the form in which the compound isadministered, the gender and age of the subject to be treated, the bodyweight of the subject to be treated, and the preference and experienceof the attending physician. The clinically-established dosages of theTILs may also be used if appropriate. The amounts of the pharmaceuticalcompositions administered using the methods herein, such as the dosagesof TILs, will be dependent on the human or mammal being treated, theseverity of the disorder or condition, the rate of administration, thedisposition of the active pharmaceutical ingredients and the discretionof the prescribing physician.

In some embodiments, TILs may be administered in a single dose. Suchadministration may be by injection, e.g., intravenous injection. In someembodiments, TILs may be administered in multiple doses. Dosing may beonce, twice, three times, four times, five times, six times, or morethan six times per year. Dosing may be once a month, once every twoweeks, once a week, or once every other day. Administration of TILs maycontinue as long as necessary.

In some embodiments, an effective dosage of TILs is about 1×10⁶, 2×10⁶,3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷,4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸,5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹,6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰,6×10^(m), 7×10^(m), 8×10¹⁰, 9×10^(m), 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹,5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, 1×10¹², 2×10¹², 3×10¹², 4×10¹²,5×10¹², 6×10¹², 7×10¹², 8×10¹², 9×10¹², 1×10¹³, 2×10¹³, 3×10¹³, 4×10¹³,5×10¹³, 6×10¹³, 7×10¹³, 8×10¹³, and 9×10¹³. In some embodiments, aneffective dosage of TILs is in the range of 1×10⁶ to 5×10⁶, 5×10⁶ to1×10⁷, 1×10⁷ to 5×10⁷, 5×10⁷ to 1×10⁸, 1×10⁸ to 5×10⁸, 5×10⁸ to 1×10⁹,1×10⁹ to 5×10⁹, 5×10⁹ to 1×10¹⁰, 1×10¹⁰ to 5×10¹⁰, 5×10¹⁰ to 1×10¹¹,5×10¹¹ to 1×10¹², 1×10¹² to 5×10¹², and 5×10¹² to 1×10¹³.

In some embodiments, an effective dosage of TILs is in the range ofabout 0.01 mg/kg to about 4.3 mg/kg, about 0.15 mg/kg to about 3.6mg/kg, about 0.3 mg/kg to about 3.2 mg/kg, about 0.35 mg/kg to about2.85 mg/kg, about 0.15 mg/kg to about 2.85 mg/kg, about 0.3 mg to about2.15 mg/kg, about 0.45 mg/kg to about 1.7 mg/kg, about 0.15 mg/kg toabout 1.3 mg/kg, about 0.3 mg/kg to about 1.15 mg/kg, about 0.45 mg/kgto about 1 mg/kg, about 0.55 mg/kg to about 0.85 mg/kg, about 0.65 mg/kgto about 0.8 mg/kg, about 0.7 mg/kg to about 0.75 mg/kg, about 0.7 mg/kgto about 2.15 mg/kg, about 0.85 mg/kg to about 2 mg/kg, about 1 mg/kg toabout 1.85 mg/kg, about 1.15 mg/kg to about 1.7 mg/kg, about 1.3 mg/kgmg to about 1.6 mg/kg, about 1.35 mg/kg to about 1.5 mg/kg, about 2.15mg/kg to about 3.6 mg/kg, about 2.3 mg/kg to about 3.4 mg/kg, about 2.4mg/kg to about 3.3 mg/kg, about 2.6 mg/kg to about 3.15 mg/kg, about 2.7mg/kg to about 3 mg/kg, about 2.8 mg/kg to about 3 mg/kg, or about 2.85mg/kg to about 2.95 mg/kg.

In some embodiments, an effective dosage of TILs is in the range ofabout 1 mg to about 500 mg, about 10 mg to about 300 mg, about 20 mg toabout 250 mg, about 25 mg to about 200 mg, about 1 mg to about 50 mg,about 5 mg to about 45 mg, about 10 mg to about 40 mg, about 15 mg toabout 35 mg, about 20 mg to about 30 mg, about 23 mg to about 28 mg,about 50 mg to about 150 mg, about 60 mg to about 140 mg, about 70 mg toabout 130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg,or about 95 mg to about 105 mg, about 98 mg to about 102 mg, about 150mg to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about230 mg, about 180 mg to about 220 mg, about 190 mg to about 210 mg,about 195 mg to about 205 mg, or about 198 to about 207 mg.

An effective amount of the TILs may be administered in either single ormultiple doses by any of the accepted modes of administration of agentshaving similar utilities, including intranasal and transdermal routes,by intra-arterial injection, intravenously, intraperitoneally,parenterally, intramuscularly, subcutaneously, topically, bytransplantation, or by inhalation.

G. Optional Cell Viability Analyses

Optionally, a cell viability assay can be performed after the Step Bfirst expansion, using standard assays known in the art. For example, atrypan blue exclusion assay can be done on a sample of the bulk TILs,which selectively labels dead cells and allows a viability assessment.Other assays for use in testing viability can include but are notlimited to the Alamar blue assay; and the MTT assay.

1. Cell Counts, Viability, Flow Cytometry

In some embodiments, cell counts and/or viability are measured. Theexpression of markers such as but not limited CD3, CD4, CD8, and CD56,as well as any other disclosed or described herein, can be measured byflow cytometry with antibodies, for example but not limited to thosecommercially available from BD Bio-sciences (BD Biosciences, San Jose,Calif.) using a FACSCanto™ flow cytometer (BD Biosciences). The cellscan be counted manually using a disposable c-chip hemocytometer (VWR,Batavia, Ill.) and viability can be assessed using any method known inthe art, including but not limited to trypan blue staining.

In some cases, the bulk TIL population can be cryopreserved immediately,using the protocols discussed below. Alternatively, the bulk TILpopulation can be subjected to REP and then cryopreserved as discussedbelow. Similarly, in the case where genetically modified TILs will beused in therapy, the bulk or REP TIL populations can be subjected togenetic modifications for suitable treatments.

2. Cell Cultures

In an embodiment, a method for expanding TILs may include using about5,000 mL to about 25,000 mL of cell medium, about 5,000 mL to about10,000 mL of cell medium, or about 5,800 mL to about 8,700 mL of cellmedium. In an embodiment, expanding the number of TILs uses no more thanone type of cell culture medium. Any suitable cell culture medium may beused, e.g., AIM-V cell medium (L-glutamine, 50 μM streptomycin sulfate,and 10 μM gentamicin sulfate) cell culture medium (Invitrogen, CarlsbadCalif.). In this regard, the inventive methods advantageously reduce theamount of medium and the number of types of medium required to expandthe number of TIL. In an embodiment, expanding the number of TIL maycomprise adding fresh cell culture media to the cells (also referred toas feeding the cells) no more frequently than every third or fourth day.Expanding the number of cells in a gas permeable container simplifiesthe procedures necessary to expand the number of cells by reducing thefeeding frequency necessary to expand the cells.

In an embodiment, the cell medium in the first and/or second gaspermeable container is unfiltered. The use of unfiltered cell medium maysimplify the procedures necessary to expand the number of cells. In anembodiment, the cell medium in the first and/or second gas permeablecontainer lacks beta-mercaptoethanol (BME).

In an embodiment, the duration of the method comprising obtaining atumor tissue sample from the mammal; culturing the tumor tissue samplein a first gas permeable container containing cell medium therein;obtaining TILs from the tumor tissue sample; expanding the number ofTILs in a second gas permeable container containing cell medium thereinusing aAPCs for a duration of about 14 to about 42 days, e.g., about 28days.

In an embodiment, TILs are expanded in gas-permeable containers.Gas-permeable containers have been used to expand TILs using PBMCs usingmethods, compositions, and devices known in the art, including thosedescribed in U.S. Patent Application Publication No. 2005/0106717 A1,the disclosures of which are incorporated herein by reference. In anembodiment, TILs are expanded in gas-permeable bags. In an embodiment,TILs are expanded using a cell expansion system that expands TILs in gaspermeable bags, such as the Xuri Cell Expansion System W25 (GEHealthcare). In an embodiment, TILs are expanded using a cell expansionsystem that expands TILs in gas permeable bags, such as the WAVEBioreactor System, also known as the Xuri Cell Expansion System W5 (GEHealthcare). In an embodiment, the cell expansion system includes a gaspermeable cell bag with a volume selected from the group consisting ofabout 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL,about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L,about 9 L, and about 10 L. In an embodiment, TILs can be expanded inG-Rex flasks (commercially available from Wilson Wolf Manufacturing).Such embodiments allow for cell populations to expand from about 5×10⁵cells/cm² to between 10×10⁶ and 30×10⁶ cells/cm². In an embodiment thisexpansion is conducted without adding fresh cell culture media to thecells (also referred to as feeding the cells). In an embodiment, this iswithout feeding so long as medium resides at a height of about 10 cm inthe G-Rex flask. In an embodiment this is without feeding but with theaddition of one or more cytokines. In an embodiment, the cytokine can beadded as a bolus without any need to mix the cytokine with the medium.Such containers, devices, and methods are known in the art and have beenused to expand TILs, and include those described in U.S. PatentApplication Publication No. US 2014/0377739A1, International PublicationNo. WO 2014/210036 A1, U.S. Patent Application Publication No. us2013/0115617 A1, International Publication No. WO 2013/188427 A1, U.S.Patent Application Publication No. US 2011/0136228 A1, U.S. Pat. No.8,809,050 B2, International publication No. WO 2011/072088 A2, U.S.Patent Application Publication No. US 2016/0208216 A1, U.S. PatentApplication Publication No. US 2012/0244133 A1, InternationalPublication No. WO 2012/129201 A1, U.S. Patent Application PublicationNo. US 2013/0102075 A1, U.S. Pat. No. 8,956,860 B2, InternationalPublication No. WO 2013/173835 A1, U.S. Patent Application PublicationNo. US 2015/0175966 A1, the disclosures of which are incorporated hereinby reference. Such processes are also described in Jin et al., J.Immunotherapy, 2012, 35:283-292. Optional Genetic Engineering of TILs

In some embodiments, the TILs are optionally genetically engineered toinclude additional functionalities, including, but not limited to, ahigh-affinity T cell receptor (TCR), e.g., a TCR targeted at atumor-associated antigen such as MAGE-1, HER2, or NY-ESO-1, or achimeric antigen receptor (CAR) which binds to a tumor-associated cellsurface molecule (e.g., mesothelin) or lineage-restricted cell surfacemolecule (e.g., CD19).

H. Optional Cryopreservation of TILs

As discussed above, and exemplified in Steps A through E as provided inFIG. 27, cryopreservation can occur at numerous points throughout theTIL expansion process. In some embodiments, the expanded population ofTILs after the second expansion (as provided for example, according toStep D of FIG. 27) can be cryopreserved. Cryopreservation can begenerally accomplished by placing the TIL population into a freezingsolution, e.g., 85% complement inactivated AB serum and 15% dimethylsulfoxide (DMSO). The cells in solution are placed into cryogenic vialsand stored for 24 hours at −80° C., with optional transfer to gaseousnitrogen freezers for cryopreservation. See Sadeghi, et al., ActaOncologica 2013, 52, 978-986. In some embodiments, the TILs arecryopreserved in 5% DMSO. In some embodiments, the TILs arecryopreserved in cell culture media plus 5% DMSO. In some embodiments,the TILs are cryopreserved according to the methods provided in Examples8 and 9.

When appropriate, the cells are removed from the freezer and thawed in a37° C. water bath until approximately ⅘ of the solution is thawed. Thecells are generally resuspended in complete media and optionally washedone or more times. In some embodiments, the thawed TILs can be countedand assessed for viability as is known in the art.

I. Phenotypic Characteristics of Expanded TILs

In some embodiment, the TILs are analyzed for expression of numerousphenotype markers after expansion, including those described herein andin the Examples. In an embodiment, expression of one or more phenotypicmarkers is examined. In some embodiments, the phenotypic characteristicsof the TILs are analyzed after the first expansion in Step B. In someembodiments, the phenotypic characteristics of the TILs are analyzedduring the transition in Step C. In some embodiments, the phenotypiccharacteristics of the TILs are analyzed during the transition accordingto Step C and after cryopreservation. In some embodiments, thephenotypic characteristics of the TILs are analyzed after the secondexpansion according to Step D. In some embodiments, the phenotypiccharacteristics of the TILs are analyzed after two or more expansionsaccording to Step D. In some embodiments, the marker is selected fromthe group consisting of TCRab (i.e., TCRα/β), CD57, CD28, CD4, CD27,CD56, CD8a, CD45RA, CD8a, CCR7, CD4, CD3, CD38, and HLA-DR. In someembodiments, the marker is selected from the group consisting of TCRab(i.e., TCRα/β), CD57, CD28, CD4, CD27, CD56, and CD8a. In an embodiment,the marker is selected from the group consisting of CD45RA, CD8a, CCR7,CD4, CD3, CD38, and HLA-DR. In some embodiments, expression of one, two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, or fourteen markers is examined. In some embodiments, theexpression from one or more markers from each group is examined. In someembodiments, one or more of HLA-DR, CD38, and CD69 expression ismaintained (i.e., does not exhibit a statistically significantdifference) in fresh TILs as compared to thawed TILs. In someembodiments, the activation status of TILs is maintained in the thawedTILs.

In an embodiment, expression of one or more regulatory markers ismeasured. In some embodiments, the regulatory marker is selected fromthe group consisting of CD137, CD8a, Lag3, CD4, CD3, PD-1, TIM-3, CD69,CD8a, TIGIT, CD4, CD3, KLRG1, and CD154. In some embodiments, theregulatory marker is selected from the group consisting of CD137, CD8a,Lag3, CD4, CD3, PD-1, and TIM-3. In some embodiments, the regulatorymarker is selected from the group consisting of CD69, CD8a, TIGIT, CD4,CD3, KLRG1, and CD154. In some embodiments, regulatory moleculeexpression is decreased in thawed TILs as compared to fresh TILs. Insome embodiments, expression of regulatory molecules LAG-3 and TIM-3 isdecreased in thawed TILs as compared to fresh TILs. In some embodiments,there is no significant difference in CD4, CD8, NK, TCRαβ expression. Insome embodiments, there is no significant difference in CD4, CD8, NK,TCRαβ expression, and/or memory markers in fresh TILs as compared tothawed TILs. In some embodiments, there is no significant difference inCD4, CD8, NK, TCRαβ expression between the TILs produced by the methodsprovided herein, as exemplified for example in FIG. 27, and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 27.

In some embodiments, no selection of the first population of TILs,second population of TILs, third population of TILs, harvested TILpopulation, and/or the therapeutic TIL population based on CD4, CD8,and/or NK, TCRαβ expression is performed during any of steps, includingthose discussed above or as provided for example in FIG. 27. In someembodiments, no selection of the first population of TILs based on CD4,CD8, and/or NK, TCRαβ is performed. In some embodiments, no selection ofthe second population of TILs based on CD4, CD8, and/or NK, TCRαβexpression is performed. In some embodiments, no selection of the thirdpopulation of TILs based on CD4, CD8, and/or NK, TCRαβ expression isperformed. In some embodiments, no selection of the harvested populationof TILs based on CD4, CD8, and/or NK, TCRαβ expression is performed. Insome embodiments, no selection of the therapeutic population of TILsbased on CD4, CD8, and/or NK, TCRαβ expression is performed.

In an embodiment, no selection of the first population of TILs, secondpopulation of TILs, third population of TILs, or harvested TILpopulation based on CD4, CD8, and/or NK, TCRαβ expression is performedduring any of steps (a) to (f) of the method for expanding tumorinfiltrating lymphocytes (TILs) into a therapeutic population of TILscomprising:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient by processing a tumor sample obtained from the        patient into multiple tumor fragments;    -   (b) adding the tumor fragments into a closed system;    -   (c) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2 to        produce a second population of TILs, wherein the first expansion        is performed in a closed container providing a first        gas-permeable surface area, wherein the first expansion is        performed for about 3-14 days to obtain the second population of        TILs, wherein the second population of TILs is at least 50-fold        greater in number than the first population of TILs, and wherein        the transition from step (b) to step (c) occurs without opening        the system;    -   (d) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        third population of TILs, wherein the second expansion is        performed for about 7-14 days to obtain the third population of        TILs, wherein the third population of TILs is a therapeutic        population of TILs which comprises an increased subpopulation of        effector T cells and/or central memory T cells relative to the        second population of TILs, wherein the second expansion is        performed in a closed container providing a second gas-permeable        surface area, and wherein the transition from step (c) to        step (d) occurs without opening the system;    -   (e) harvesting the therapeutic population of TILs obtained from        step (d), wherein the transition from step (d) to step (e)        occurs without opening the system; and    -   (f) transferring the harvested TIL population from step (e) to        an infusion bag, wherein the transfer from step (e) to (f)        occurs without opening the system.

In an embodiment, no selection of the first population of TILs, secondpopulation of TILs, third population of TILs, or harvested TILpopulation based on CD4, CD8, and/or NK, TCRαβ expression is performedduring any of steps (a) to (h) of the method for treating a subject withcancer, the method comprising administering expanded tumor infiltratinglymphocytes (TILs) comprising:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a subject by processing a tumor sample obtained from the        patient into multiple tumor fragments;    -   (b) adding the tumor fragments into a closed system;    -   (c) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2 to        produce a second population of TILs, wherein the first expansion        is performed in a closed container providing a first        gas-permeable surface area, wherein the first expansion is        performed for about 3-14 days to obtain the second population of        TILs, wherein the second population of TILs is at least 50-fold        greater in number than the first population of TILs, and wherein        the transition from step (b) to step (c) occurs without opening        the system;    -   (d) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        third population of TILs, wherein the second expansion is        performed for about 7-14 days to obtain the third population of        TILs, wherein the third population of TILs is a therapeutic        population of TILs which comprises an increased subpopulation of        effector T cells and/or central memory T cells relative to the        second population of TILs, wherein the second expansion is        performed in a closed container providing a second gas-permeable        surface area, and wherein the transition from step (c) to        step (d) occurs without opening the system;    -   (e) harvesting the therapeutic population of TILs obtained from        step (d), wherein the transition from step (d) to step (e)        occurs without opening the system; and    -   (f) transferring the harvested TIL population from step (e) to        an infusion bag, wherein the transfer from step (e) to (f)        occurs without opening the system;    -   (g) optionally cryopreserving the infusion bag comprising the        harvested TIL population from step (f) using a cryopreservation        process; and    -   (h) administering a therapeutically effective dosage of the        third population of TILs from the infusion bag in step (g) to        the patient.

In some embodiments the memory marker is selected from the groupconsisting of CCR7 and CD62L

In some embodiments, the viability of the fresh TILs as compared to thethawed TILs is at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, or at least 98%. In some embodiments, theviability of both the fresh and thawed TILs is greater than 70%, greaterthan 75%, greater than 80%, greater than 85%, greater than 90%, greaterthan 95%, or greater than 98%. In some embodiments, the viability ofboth the fresh and thawed product is greater than 80%, greater than 81%,greater than 82%, greater than 83%, greater than 84%, greater than 85%,greater than 86%, greater than 87%, greater than 88%, greater than 89%,or greater than 90%. In some embodiments, the viability of both thefresh and thawed product is greater than 86%.

In an embodiment, restimulated TILs can also be evaluated for cytokinerelease, using cytokine release assays. In some embodiments, TILs can beevaluated for interferon-7 (IFN-7) secretion in response to stimulationeither with OKT3 or co-culture with autologous tumor digest. Forexample, in embodiments employing OKT3 stimulation, TILs are washedextensively, and duplicate wells are prepared with 1×10⁵ cells in 0.2 mLCM in 96-well flat-bottom plates precoated with 0.1 or 1.0 μg/mL of OKT3diluted in phosphate-buffered saline. After overnight incubation, thesupernatants are harvested and IFN-7 in the supernatant is measured byELISA (Pierce/Endogen, Woburn, Mass.). For the co-culture assay, 1×10⁵TIL cells are placed into a 96-well plate with autologous tumor cells.(1:1 ratio). After a 24-hour incubation, supernatants are harvested andIFN-7 release can be quantified, for example by ELISA.

Flow cytometric analysis of cell surface biomarkers: TIL samples werealiquoted for flow cytometric analysis of cell surface markers see, forExample see Examples 7, 8, and 9.

In some embodiments, the TILs are being evaluated for various regulatorymarkers. In some embodiments, the regulatory marker is selected from thegroup consisting of TCR α/β, CD56, CD27, CD28, CD57, CD45RA, CD45RO,CD25, CD127, CD95, IL-2R−, CCR7, CD62L, KLRG1, and CD122. In someembodiments, the regulatory marker is TCR α/β. In some embodiments, theregulatory marker is CD56. In some embodiments, the regulatory marker isCD27. In some embodiments, the regulatory marker is CD28. In someembodiments, the regulatory marker is CD57. In some embodiments, theregulatory marker is CD45RA. In some embodiments, the regulatory markeris CD45RO. In some embodiments, the regulatory marker is CD25. In someembodiments, the regulatory marker is CD127. In some embodiments, theregulatory marker is CD95. In some embodiments, the regulatory marker isIL-2R−. In some embodiments, the regulatory marker is CCR7. In someembodiments, the regulatory marker is CD62L. In some embodiments, theregulatory marker is KLRG1. In some embodiments, the regulatory markeris CD122.

In an embodiment, the expanded TILs are analyzed for expression ofnumerous phenotype markers, including those described herein and in theExamples. In an embodiment, expression of one or more phenotypic markersis examined. In some embodiments, the marker is selected from the groupconsisting of TCRab (i.e., TCRα/β), CD57, CD28, CD4, CD27, CD56, CD8a,CD45RA, CD8a, CCR7, CD4, CD3, CD38, and HLA-DR. In some embodiments, themarker is selected from the group consisting of TCRab (i.e., TCRα/β),CD57, CD28, CD4, CD27, CD56, and CD8a. In an embodiment, the marker isselected from the group consisting of CD45RA, CD8a, CCR7, CD4, CD3,CD38, and HLA-DR. In some embodiments, expression of one, two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, orfourteen markers is examined. In some embodiments, the expression fromone or more markers from each group is examined. In some embodiments,one or more of HLA-DR, CD38, and CD69 expression is maintained (i.e.,does not exhibit a statistically significant difference) in fresh TILsas compared to thawed TILs. In some embodiments, the activation statusof TILs is maintained in the thawed TILs.

In an embodiment, expression of one or more regulatory markers ismeasured. In some embodiments, the regulatory marker is selected fromthe group consisting of CD137, CD8a, Lag3, CD4, CD3, PD1, TIM-3, CD69,CD8a, TIGIT, CD4, CD3, KLRG1, and CD154. In some embodiments, theregulatory marker is selected from the group consisting of CD137, CD8a,Lag3, CD4, CD3, PD1, and TIM-3. In some embodiments, the regulatorymarker is selected from the group consisting of CD69, CD8a, TIGIT, CD4,CD3, KLRG1, and CD154. In some embodiments, regulatory moleculeexpression is decreased in thawed TILs as compared to fresh TILs. Insome embodiments, expression of regulatory molecules LAG-3 and TIM-3 isdecreased in thawed TILs as compared to fresh TILs. In some embodiments,there is no significant difference in CD4, CD8, NK, TCRαβ expression. Insome embodiments, there is no significant difference in CD4, CD8, NK,TCRαβ expression, and/or memory markers in fresh TILs as compared tothawed TILs.

In some embodiments the memory marker is selected from the groupconsisting of CCR7 and CD62L.

In some embodiments, the viability of the fresh TILs as compared to thethawed TILs is at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, or at least 98%. In some embodiments, theviability of both the fresh and thawed TILs is greater than 70%, greaterthan 75%, greater than 80%, greater than 85%, greater than 90%, greaterthan 95%, or greater than 98%. In some embodiments, the viability ofboth the fresh and thawed product is greater than 80%, greater than 81%,greater than 82%, greater than 83%, greater than 84%, greater than 85%,greater than 86%, greater than 87%, greater than 88%, greater than 89%,or greater than 90%. In some embodiments, the viability of both thefresh and thawed product is greater than 86%.

In an embodiment, restimulated TILs can also be evaluated for cytokinerelease, using cytokine release assays. In some embodiments, TILs can beevaluated for interferon-7 (IFN-7) secretion in response to stimulationeither with OKT3 or coculture with autologous tumor digest. For example,in embodiments employing OKT3 stimulation, TILs are washed extensively,and duplicate wells are prepared with 1×10⁵ cells in 0.2 mL CM in96-well flat-bottom plates precoated with 0.1 or 1.0 μg/mL of OKT3diluted in phosphate-buffered saline. After overnight incubation, thesupernatants are harvested and IFN-7 in the supernatant is measured byELISA (Pierce/Endogen, Woburn, Mass.). For the coculture assay, 1×10⁵TIL cells are placed into a 96-well plate with autologous tumor cells.(1:1 ratio). After a 24-hour incubation, supernatants are harvested andIFN-7 release can be quantified, for example by ELISA.

In some embodiments, the phenotypic characterization is examined aftercryopreservation.

J. Metabolic Health of Expanded TILs

The restimulated TILs are characterized by significant enhancement ofbasal glycolysis as compared to either freshly harvested TILs and/orpost-thawed TILs. In an embodiment, no selection of the first populationof TILs, second population of TILs, third population of TILs, harvestedTIL population, and/or the therapeutic TIL population based on CD8expression is performed during any of steps, including those discussedabove or as provided for example in FIG. 27. In some embodiments, noselection of the first population of TILs based on CD8 expression isperformed. In some embodiments, no selection of the second population ofTILs based on CD8 expression is performed. In some embodiments, noselection of the third population of TILs based on CD8 expression isperformed. In some embodiments, no selection of the harvested populationof TILs based on CD8 expression is performed. In some embodiments, noselection of the therapeutic population of TILs based on CD8 expressionis performed.

In an embodiment, no selection of the first population of TILs, secondpopulation of TILs, third population of TILs, or harvested TILpopulation based on CD8 expression is performed during any of steps (a)to (f) of the method for expanding tumor infiltrating lymphocytes (TILs)into a therapeutic population of TILs comprising:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient by processing a tumor sample obtained from the        patient into multiple tumor fragments;    -   (b) adding the tumor fragments into a closed system;    -   (c) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2 to        produce a second population of TILs, wherein the first expansion        is performed in a closed container providing a first        gas-permeable surface area, wherein the first expansion is        performed for about 3-14 days to obtain the second population of        TILs, wherein the second population of TILs is at least 50-fold        greater in number than the first population of TILs, and wherein        the transition from step (b) to step (c) occurs without opening        the system;    -   (d) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        third population of TILs, wherein the second expansion is        performed for about 7-14 days to obtain the third population of        TILs, wherein the third population of TILs is a therapeutic        population of TILs which comprises an increased subpopulation of        effector T cells and/or central memory T cells relative to the        second population of TILs, wherein the second expansion is        performed in a closed container providing a second gas-permeable        surface area, and wherein the transition from step (c) to        step (d) occurs without opening the system;    -   (e) harvesting the therapeutic population of TILs obtained from        step (d), wherein the transition from step (d) to step (e)        occurs without opening the system; and    -   (f) transferring the harvested TIL population from step (e) to        an infusion bag, wherein the transfer from step (e) to (f)        occurs without opening the system.

In an embodiment, no selection of the first population of TILs, secondpopulation of TILs, third population of TILs, or harvested TILpopulation based on CD8 expression is performed during any of steps (a)to (h) of the method for treating a subject with cancer, the methodcomprising administering expanded tumor infiltrating lymphocytes (TILs)comprising:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a subject by processing a tumor sample obtained from the        patient into multiple tumor fragments;    -   (b) adding the tumor fragments into a closed system;    -   (c) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2 to        produce a second population of TILs, wherein the first expansion        is performed in a closed container providing a first        gas-permeable surface area, wherein the first expansion is        performed for about 3-14 days to obtain the second population of        TILs, wherein the second population of TILs is at least 50-fold        greater in number than the first population of TILs, and wherein        the transition from step (b) to step (c) occurs without opening        the system;    -   (d) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        third population of TILs, wherein the second expansion is        performed for about 7-14 days to obtain the third population of        TILs, wherein the third population of TILs is a therapeutic        population of TILs which comprises an increased subpopulation of        effector T cells and/or central memory T cells relative to the        second population of TILs, wherein the second expansion is        performed in a closed container providing a second gas-permeable        surface area, and wherein the transition from step (c) to        step (d) occurs without opening the system;    -   (e) harvesting the therapeutic population of TILs obtained from        step (d), wherein the transition from step (d) to step (e)        occurs without opening the system; and    -   (f) transferring the harvested TIL population from step (e) to        an infusion bag, wherein the transfer from step (e) to (f)        occurs without opening the system;    -   (g) optionally cryopreserving the infusion bag comprising the        harvested TIL population from step (f) using a cryopreservation        process; and    -   (h) administering a therapeutically effective dosage of the        third population of TILs from the infusion bag in step (g) to        the patient.

The TILs prepared by the methods described herein are characterized bysignificant enhancement of basal glycolysis as compared to, for example,freshly harvested TILs and/or TILs prepared using other methods thanthose provide herein including for example, methods other than thoseembodied in FIG. 27. In an embodiment, no selection of the firstpopulation of TILs, second population of TILs, third population of TILs,harvested TIL population, and/or the therapeutic TIL population based onCD8 expression is performed during any of steps, including thosediscussed above or as provided for example in FIG. 27. In someembodiments, no selection of the first population of TILs based on CD8expression is performed. In some embodiments, no selection of the secondpopulation of TILs based on CD8 expression is performed. In someembodiments, no selection of the third population of TILs based on CD8expression is performed. In some embodiments, no selection of theharvested population of TILs based on CD8 expression is performed. Insome embodiments, no selection of the therapeutic population of TILsbased on CD8 expression is performed. In an embodiment, no selection ofthe first population of TILs, second population of TILs, thirdpopulation of TILs, or harvested TIL population based on CD8 expressionis performed during any of steps (a) to (h).

Spare respiratory capacity (SRC) and glycolytic reserve can be evaluatedfor TILs expanded with different methods of the present disclosure. TheSeahorse XF Cell Mito Stress Test measures mitochondrial function bydirectly measuring the oxygen consumption rate (OCR) of cells, usingmodulators of respiration that target components of the electrontransport chain in the mitochondria. The test compounds (oligomycin,FCCP, and a mix of rotenone and antimycin A, described below) areserially injected to measure ATP production, maximal respiration, andnon-mitochondrial respiration, respectively. Proton leak and sparerespiratory capacity are then calculated using these parameters andbasal respiration. Each modulator targets a specific component of theelectron transport chain. Oligomycin inhibits ATP synthase (complex V)and the decrease in OCR following injection of oligomycin correlates tothe mitochondrial respiration associated with cellular ATP production.Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) is anuncoupling agent that collapses the proton gradient and disrupts themitochondrial membrane potential. As a result, electron flow through theelectron transport chain is uninhibited and oxygen is maximally consumedby complex IV. The FCCP-stimulated OCR can then be used to calculatespare respiratory capacity, defined as the difference between maximalrespiration and basal respiration. Spare respiratory capacity (SRC) is ameasure of the ability of the cell to respond to increased energydemand. The third injection is a mix of rotenone, a complex I inhibitor,and antimycin A, a complex III inhibitor. This combination shuts downmitochondrial respiration and enables the calculation ofnonmitochondrial respiration driven by processes outside themitochondria. In some embodiments, the comparison is to, for example,freshly harvested TILs and/or TILs prepared using other methods thanthose provide herein including for example, methods other than thoseembodied in FIG. 27.

In some embodiments, the metabolic assay is basal respiration. Ingeneral, second expansion TILs have a basal respiration rate that is atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 97%, at least 98%, or at least 99% of the basal respiration rateof freshly harvested TILs and/or TILs prepared using other methods thanthose provide herein including for example, methods other than thoseembodied in FIG. 27. In some embodiments, the basal respiration rate isfrom about 50% to about 99% of the basal respiration rate of freshlyharvested TILs and/or TILs prepared using other methods than thoseprovide herein including for example, methods other than those embodiedin FIG. 27. In some embodiments, the basal respiration rate is fromabout 60% to about 99% of the basal respiration rate of freshlyharvested TILs and/or TILs prepared using other methods than thoseprovide herein including for example, methods other than those embodiedin FIG. 27. In some embodiments, the basal respiration rate is fromabout 70% to about 99% of the basal respiration rate of freshlyharvested TILs and/or TILs prepared using other methods than thoseprovide herein including for example, methods other than those embodiedin FIG. 27. In some embodiments, the basal respiration rate is fromabout 80% to about 99% of the basal respiration rate of freshlyharvested TILs and/or TILs prepared using other methods than thoseprovide herein including for example, methods other than those embodiedin FIG. 27. In some embodiments, the basal respiration rate is fromabout 90% to about 99% of the basal respiration rate of freshlyharvested TILs and/or TILs prepared using other methods than thoseprovide herein including for example, methods other than those embodiedin FIG. 27. In some embodiments, the basal respiration rate is fromabout 95% to about 99% of the basal respiration rate of freshlyharvested TILs and/or TILs prepared using other methods than thoseprovide herein including for example, methods other than those embodiedin FIG. 27. In some embodiments, the second expansion TILs or secondadditional expansion TILs (such as, for example, those described in StepD of FIG. 27, including TILs referred to as reREP TILs) have a basalrespiration rate that is not statistically significantly different thanthe basal respiration rate of freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 27. In someembodiments, the comparison is to, for example, freshly harvested TILsand/or TILs prepared using other methods than those provide hereinincluding for example, methods other than those embodied in FIG. 27.

In some embodiments, the metabolic assay is spare respiratory capacity.In general, the second expansion TILs or second additional expansionTILs (such as, for example, those described in Step D of FIG. 27,including TILs referred to as reREP TILs) have a spare respiratorycapacity that is at least is at least 50%, at least 55%, at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 97%, at least 98%, or at least 99% ofthe basal respiration rate of freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 27. In someembodiments, the spare respiratory capacity is from about 50% to about99% of the basal respiration rate of freshly harvested TILs. In someembodiments, the spare respiratory capacity is from about 50% to about99% of the basal respiration rate of freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 27. In someembodiments, the spare respiratory capacity is from about 60% to about99% of the basal respiration rate of freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 27. In someembodiments, the spare respiratory capacity is from about 70% to about99% of the basal respiration rate of freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 27. In someembodiments, the spare respiratory capacity is from about 80% to about99% of the basal respiration rate of freshly harvested TILs. In someembodiments, the spare respiratory capacity is from about 90% to about99% of the basal respiration rate of freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 27. In someembodiments, the spare respiratory capacity is from about 95% to about99% of the basal respiration rate of freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 27. In someembodiments, the second expansion TILs or second additional expansionTILs (such as, for example, those described in Step D of FIG. 27,including TILs referred to as reREP TILs) have a spare respiratorycapacity that is not statistically significantly different than thebasal respiration rate of freshly harvested TILs and/or TILs preparedusing other methods than those provide herein including for example,methods other than those embodied in FIG. 27.

In general, second expansion TILs or second additional expansion TILs(such as, for example, those described in Step D of FIG. 27, includingTILs referred to as reREP TILs) have a spare respiratory capacity thatis at least is at least 50%, at least 55%, at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 97%, at least 98%, or at least 99% of the basalrespiration rate of freshly harvested TILs and/or TILs prepared usingother methods than those provide herein including for example, methodsother than those embodied in FIG. 27. In some embodiments, the metabolicassay measured is glycolytic reserve. In some embodiments, the metabolicassay is spare respiratory capacity. To measure cellular (respiratory)metabolism cells were treated with inhibitors of mitochondrialrespiration and glycolysis to determine a metabolic profile for the TILconsisting of the following measures: baseline oxidative phosphorylation(as measured by OCR), spare respiratory capacity, baseline glycolyticactivity (as measured by ECAR), and glycolytic reserve. Metabolicprofiles were performed using the Seahorse CombinationMitochondrial/Glycolysis Stress Test Assay (including the kitcommercially available from Agilent®), which allows for determining acells' capacity to perform glycolysis upon blockage of mitochondrial ATPproduction. In some embodiments, cells are starved of glucose, thenglucose is injected, followed by a stress agent. In some embodiments,the stress agent is selected from the group consisting of oligomycin,FCCP, rotenone, antimycin A and/or 2-deoxyglucose (2-DG), as well ascombinations thereof. In some embodiments, oligomycin is added at 10 mM.In some embodiments, FCCP is added at 10 mM. In some embodiments,rotenone is added at 2.5 mM. In some embodiments, antimycin A is addedat 2.5 mM. In some embodiments, 2-deoxyglucose (2-DG) is added at 500mM. In some embodiments, glycolytic capacity, glycolytic reserve, and/ornon-glycolytic acidification are measured. In general, TILs have aglycolytic reserve that is at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 97%, at least 98%, or at least 99% ofthe basal respiration rate of freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 27. In someembodiments, the glycolytic reserve is from about 50% to about 99% ofthe basal respiration rate of freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 27. In someembodiments, the glycolytic reserve is from about 60% to about 99% ofthe basal respiration rate of freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 27. In someembodiments, the glycolytic reserve is from about 70% to about 99% ofthe basal respiration rate of freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 27. In someembodiments, the glycolytic reserve is from about 80% to about 99% ofthe basal respiration rate of freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 27. In someembodiments, the glycolytic reserve is from about 90% to about 99% ofthe basal respiration rate of freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 27. In someembodiments, the glycolytic reserve is from about 95% to about 99% ofthe basal respiration rate of freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 27.

In some embodiments, the metabolic assay is basal glycolysis. In someembodiments, second expansion TILs or second additional expansion TILs(such as, for example, those described in Step D of FIG. 27, includingTILs referred to as reREP TILs) have an increase in basal glycolysis ofat least two-fold, at least three-fold, at least four-fold, at leastfive-fold, at least six-fold, at least 7-fold, at least eight-fold, atleast nine-fold, or at least ten-fold as compared to freshly harvestedTILs and/or TILs prepared using other methods than those provide hereinincluding for example, methods other than those embodied in FIG. 27. Insome embodiments, the second expansion TILs or second additionalexpansion TILs (such as, for example, those described in Step D of FIG.27, including TILs referred to as reREP TILs) have an increase in basalglycolysis of about two-fold to about ten-fold as compared to freshlyharvested TILs and/or TILs prepared using other methods than thoseprovide herein including for example, methods other than those embodiedin FIG. 27. In some embodiments, the second expansion TILs or secondadditional expansion TILs (such as, for example, those described in StepD of FIG. 27, including TILs referred to as reREP TILs) have an increasein basal glycolysis of about two-fold to about eight-fold as compared tofreshly harvested TILs and/or TILs prepared using other methods thanthose provide herein including for example, methods other than thoseembodied in FIG. 27. In some embodiments, second expansion TILs orsecond additional expansion TILs (such as, for example, those describedin Step D of FIG. 27, including TILs referred to as reREP TILs) have anincrease in basal glycolysis of about three-fold to about seven-fold ascompared to freshly harvested TILs and/or TILs prepared using othermethods than those provide herein including for example, methods otherthan those embodied in FIG. 27. In some embodiments, the secondexpansion TILs or second additional expansion TILs (such as, forexample, those described in Step D of FIG. 27, including TILs referredto as reREP TILs) have an increase in basal glycolysis of about two-foldto about four-fold as compared to freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 27. In someembodiments, the second expansion TILs or second additional expansionTILs (such as, for example, those described in Step D of FIG. 27,including TILs referred to as reREP TILs) have an increase in basalglycolysis of about two-fold to about three-fold as compared to freshlyharvested TILs and/or TILs prepared using other methods than thoseprovide herein including for example, methods other than those embodiedin FIG. 27.

In general, the second expansion TILs or second additional expansionTILs (such as, for example, those described in Step D of FIG. 27,including TILs referred to as reREP TILs) have a glycolytic reserve thatis at least 50%, at least 55%, at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 97%, at least 98%, or at least 99% of the basal respiration rateof freshly harvested TILs and/or TILs prepared using other methods thanthose provide herein including for example, methods other than thoseembodied in FIG. 27. In some embodiments, the glycolytic reserve is fromabout 50% to about 99% of the basal respiration rate of freshlyharvested TILs. In some embodiments, the glycolytic reserve is fromabout 60% to about 99% of the basal respiration rate of freshlyharvested TILs and/or TILs prepared using other methods than thoseprovide herein including for example, methods other than those embodiedin FIG. 27. In some embodiments, the glycolytic reserve is from about70% to about 99% of the basal respiration rate of freshly harvested TILsand/or TILs prepared using other methods than those provide hereinincluding for example, methods other than those embodied in FIG. 27. Insome embodiments, the glycolytic reserve is from about 80% to about 99%of the basal respiration rate of freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 27. In someembodiments, the glycolytic reserve is from about 90% to about 99% ofthe basal respiration rate of freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 27. In someembodiments, the glycolytic reserve is from about 95% to about 99% ofthe basal respiration rate of freshly harvested TILs.

Granzyme B Production: Granzyme B is another measure of the ability ofTIL to kill target cells. Media supernatants restimulated as describedabove using antibodies to CD3, CD28, and CD137/4-1BB were also evaluatedfor their levels of Granzyme B using the Human Granzyme B DuoSet ELISAKit (R & D Systems, Minneapolis, Minn.) according to the manufacturer'sinstructions. In some embodiments, the second expansion TILs or secondadditional expansion TILs (such as, for example, those described in StepD of FIG. 27, including TILs referred to as reREP TILs) have increasedGranzyme B production. In some embodiments, the second expansion TILs orsecond additional expansion TILs (such as, for example, those describedin Step D of FIG. 27, including TILs referred to as reREP TILs) haveincreased cytotoxic activity.

In some embodiments, telomere length can be used as a measure of cellviability and/or cellular function. In some embodiments, the telomeresare surprisingly the same length in the TILs produced by the presentinvention as compared to TILs prepared using other methods than thoseprovide herein including for example, methods other than those embodiedin FIG. 27. Telomere length measurement: Diverse methods have been usedto measure the length of telomeres in genomic DNA and cytologicalpreparations. The telomere restriction fragment (TRF) analysis is thegold standard to measure telomere length (de Lange et al., 1990).However, the major limitation of TRF is the requirement of a largeamount of DNA (1.5{circumflex over ( )}g). Two widely used techniquesfor the measurement of telomere lengths namely, fluorescence in situhybridization (FISH; Agilent Technologies, Santa Clara, Calif.) andquantitative PCR can be employed with the present invention. In someembodiments, there is no change in telomere length between the initiallyharvest TILs in Step A and the expanded TILs from for example Step D asprovided in FIG. 27.

In some embodiments, TIL health is measured by IFN-gamma (IFN-γ)secretion. In some embodiments, IFN-γ secretion is indicative of activeTILs. In some embodiments, a potency assay for IFN-γ production isemployed. IFN-γ production is another measure of cytotoxic potential.IFN-γ production can be measured by determining the levels of thecytokine IFN-γ in the media of TIL stimulated with antibodies to CD3,CD28, and CD137/4-1BB. IFN-γ levels in media from these stimulated TILcan be determined using by measuring IFN-γ release. In some embodiments,an increase in IFN-γ production in for example Step D as provided inFIG. 27 TILs as compared to initially harvested TILs in for example StepA as provided in FIG. 27 is indicative of an increase in cytotoxicpotential of the Step D TILs. In some embodiments, IFN-γ secretion isincreased one-fold, two-fold, three-fold, four-fold, or five-fold ormore. In some embodiments, IFN-γ secretion is increased one-fold. Insome embodiments, IFN-γ secretion is increased two-fold. In someembodiments, IFN-γ secretion is increased three-fold. In someembodiments, IFN-γ secretion is increased four-fold. In someembodiments, IFN-γ secretion is increased five-fold. In someembodiments, IFN-γ is measured using a Quantikine ELISA kit. In someembodiments, IFN-γ is measured in TILs ex vivo. In some embodiments,IFN-γ is measured in TILs ex vivo, including TILs produced by themethods of the present invention, including for example FIG. 27 methods,as well as freshly harvested TILs or those TILs produced by othermethods, such as those provided for example in FIG. 83 (such as forexample process 1C TILs).

In some embodiments, the cytotoxic potential of TIL to lyse target cellswas assessed using a co-culture assay of TIL with the bioluminescentcell line, P815 (Clone G6), according to a bioluminescent redirectedlysis assay (potency assay) for TIL assay which measures TILcytotoxicity in a highly sensitive dose dependent manner.

In some embodiments, the present methods provide an assay for assessingTIL viability, using the methods as described above. In someembodiments, the TILs are expanded as discussed above, including forexample as provided in FIG. 27. In some embodiments, the TILs arecryopreserved prior to being assessed for viability. In someembodiments, the viability assessment includes thawing the TILs prior toperforming a first expansion, a second expansion, and an additionalsecond expansion. In some embodiments, the present methods provide anassay for assessing cell proliferation, cell toxicity, cell death,and/or other terms related to viability of the TIL population. Viabilitycan be measured by any of the TIL metabolic assays described above aswell as any methods know for assessing cell viability that are known inthe art. In some embodiments, the present methods provide as assay forassessment of cell proliferation, cell toxicity, cell death, and/orother terms related to viability of the TILs expanded using the methodsdescribed herein, including those exemplified in FIG. 27.

The present invention also provides assay methods for determining TILviability. In some embodiments, the TILs have equal viability ascompared to freshly harvested TILs and/or TILs prepared using othermethods than those provide herein including for example, methods otherthan those embodied in FIG. 27. In some embodiments, the TILs haveincreased viability as compared to freshly harvested TILs and/or TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 27. The presentdisclosure provides methods for assaying TILs for viability by expandingtumor infiltrating lymphocytes (TILs) into a larger population of TILscomprising:

-   -   (i) obtaining a first population of TILs which has been        previously expanded;    -   (ii) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2 to        produce a second population of TILs; and    -   (iii) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        third population of TILs, wherein the third population of TILs        is at least 100-fold greater in number than the second        population of TILs, and wherein the second expansion is        performed for at least 14 days in order to obtain the third        population of TILs, wherein the third population of TILs        comprises an increased subpopulation of effector T cells and/or        central memory T cells relative to the second population of        TILs, and wherein the third population is further assayed for        viability.

In some embodiments, the method further comprises:

-   -   (iv) performing an additional second expansion by supplementing        the cell culture medium of the third population of TILs with        additional IL-2, additional OKT-3, and additional APCs, wherein        the additional second expansion is performed for at least 14        days to obtain a larger population of TILs than obtained in step        (iii), wherein the larger population of TILs comprises an        increased subpopulation of effector T cells and/or central        memory T cells relative to the third population of TILs, and        wherein the third population is further assayed for viability.

In some embodiments, prior to step (i), the cells are cryopreserved.

In some embodiments, the cells are thawed prior to performing step (i).

In some embodiments, step (iv) is repeated one to four times in order toobtain sufficient TILs for analysis.

In some embodiments, steps (i) through (iii) or (iv) are performedwithin a period of about 40 days to about 50 days.

In some embodiments, steps (i) through (iii) or (iv) are performedwithin a period of about 42 days to about 48 days.

In some embodiments, steps (i) through (iii) or (iv) are performedwithin a period of about 42 days to about 45 days.

In some embodiments, steps (i) through (iii) or (iv) are performedwithin about 44 days.

In some embodiments, the cells from steps (iii) or (iv) express CD4,CD8, and TCR αβ at levels similar to freshly harvested cells.

In some embodiments, the antigen presenting cells are peripheral bloodmononuclear cells (PBMCs).

In some embodiments, the PBMCs are added to the cell culture on any ofdays 9 through 17 in step (iii).

In some embodiments, the effector T cells and/or central memory T cellsin the larger population of TILs in step (iv) exhibit one or morecharacteristics selected from the group consisting of expression ofCD27, expression of CD28, longer telomeres, increased CD57 expression,and decreased CD56 expression, relative to effector T cells, and/orcentral memory T cells in the third population of cells.

In some embodiments, the effector T cells and/or central memory T cellsexhibit increased CD57 expression and decreased CD56 expression.

In some embodiments, the APCs are artificial APCs (aAPCs).

In some embodiments, the method further comprises the step oftransducing the first population of TILs with an expression vectorcomprising a nucleic acid encoding a high-affinity T cell receptor.

In some embodiments, the step of transducing occurs before step (i).

In some embodiments, the method further comprises the step oftransducing the first population of TILs with an expression vectorcomprising a nucleic acid encoding a chimeric antigen receptor (CAR)comprising a single chain variable fragment antibody fused with at leastone endodomain of a T-cell signaling molecule.

In some embodiments, the step of transducing occurs before step (i).

In some embodiments, the TILs are assayed for viability.

In some embodiments, the TILs are assayed for viability aftercryopreservation.

In some embodiments, the TILs are assayed for viability aftercryopreservation and after step (iv).

The diverse antigen receptors of T and B lymphocytes are produced bysomatic recombination of a limited, but large number of gene segments.These gene segments: V (variable), D (diversity), J (joining), and C(constant), determine the binding specificity and downstreamapplications of immunoglobulins and T-cell receptors (TCRs). The presentinvention provides a method for generating TILs which exhibit andincrease the T-cell repertoire diversity (sometimes referred to aspolyclonality). In some embodiments, the increase in T-cell repertoirediversity is as compared to freshly harvested TILs and/or TILs preparedusing other methods than those provide herein including for example,methods other than those embodied in FIG. 27. In some embodiments, theTILs obtained by the present method exhibit an increase in the T-cellrepertoire diversity. In some embodiments, the TILs obtained in thefirst expansion exhibit an increase in the T-cell repertoire diversity.In some embodiments, the increase in diversity is an increase in theimmunoglobulin diversity and/or the T-cell receptor diversity. In someembodiments, the diversity is in the immunoglobulin is in theimmunoglobulin heavy chain. In some embodiments, the diversity is in theimmunoglobulin is in the immunoglobulin light chain. In someembodiments, the diversity is in the T-cell receptor. In someembodiments, the diversity is in one of the T-cell receptors selectedfrom the group consisting of alpha, beta, gamma, and delta receptors. Insome embodiments, there is an increase in the expression of T-cellreceptor (TCR) alpha and/or beta. In some embodiments, there is anincrease in the expression of T-cell receptor (TCR) alpha. In someembodiments, there is an increase in the expression of T-cell receptor(TCR) beta. In some embodiments, there is an increase in the expressionof TCRab (i.e., TCRα/β).

According to the present disclosure, a method for assaying TILs forviability and/or further use in administration to a subject. In someembodiments, the method for assay tumor infiltrating lymphocytes (TILs)comprises:

-   -   (i) obtaining a first population of TILs;    -   (ii) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2 to        produce a second population of TILs; and    -   (iii) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        third population of TILs, wherein the third population of TILs        is at least 50-fold greater in number than the second population        of TILs;    -   (iv) harvesting, washing, and cryopreserving the third        population of TILs;    -   (v) storing the cryopreserved TILs at a cryogenic temperature;    -   (vi) thawing the third population of TILs to provide a thawed        third population of TILs; and    -   (vii) performing an additional second expansion of a portion of        the thawed third population of TILs by supplementing the cell        culture medium of the third population with IL-2, OKT-3, and        APCs for an additional expansion period (sometimes referred to        as a reREP period) of at least 3 days, wherein the third        expansion is performed to obtain a fourth population of TILs,        wherein the number of TILs in the fourth population of TILs is        compared to the number of TILs in the third population of TILs        to obtain a ratio;    -   (viii) determining based on the ratio in step (vii) whether the        thawed population of TILs is suitable for administration to a        patient;    -   (ix) administering a therapeutically effective dosage of the        thawed third population of TILs to the patient when the ratio of        the number of TILs in the fourth population of TILs to the        number of TILs in the third population of TILs is determined to        be greater than 5:1 in step (viii).

In some embodiments, the additional expansion period (sometimes referredto as a reREP period) is performed until the ratio of the number of TILsin the fourth population of TILs to the number of TILs in the thirdpopulation of TILs is greater than 50:1.

In some embodiments, the number of TILs sufficient for a therapeuticallyeffective dosage is from about 2.3×10¹⁰ to about 13.7×10¹⁰.

In some embodiments, steps (i) through (vii) are performed within aperiod of about 40 days to about 50 days. In some embodiments, steps (i)through (vii) are performed within a period of about 42 days to about 48days. In some embodiments, steps (i) through (vii) are performed withina period of about 42 days to about 45 days. In some embodiments, steps(i) through (vii) are performed within about 44 days.

In some embodiments, the cells from steps (iii) or (vii) express CD4,CD8, and TCR αβ at levels similar to freshly harvested cells. In someembodiments the cells are TILs.

In some embodiments, the antigen presenting cells are peripheral bloodmononuclear cells (PBMCs). In some embodiments, the PBMCs are added tothe cell culture on any of days 9 through 17 in step (iii).

In some embodiments, the effector T cells and/or central memory T cellsin the larger population of TILs in steps (iii) or (vii) exhibit one ormore characteristics selected from the group consisting of expression ofCD27, expression of CD28, longer telomeres, increased CD57 expression,and decreased CD56 expression, relative to effector T cells, and/orcentral memory T cells in the third population of cells.

In some embodiments, the effector T cells and/or central memory T cellsexhibit increased CD57 expression and decreased CD56 expression.

In some embodiments, the APCs are artificial APCs (aAPCs).

In some embodiments, the step of transducing the first population ofTILs with an expression vector comprising a nucleic acid encoding ahigh-affinity T cell receptor.

In some embodiments, the step of transducing occurs before step (i).

In some embodiments, the step of transducing the first population ofTILs with an expression vector comprising a nucleic acid encoding achimeric antigen receptor (CAR) comprising a single chain variablefragment antibody fused with at least one endodomain of a T-cellsignaling molecule.

In some embodiments, the step of transducing occurs before step (i).

In some embodiments, the TILs are assayed for viability after step(vii).

The present disclosure also provides further methods for assaying TILs.In some embodiments, the disclosure provides a method for assaying TILscomprising:

-   -   (i) obtaining a portion of a first population of cryopreserved        TILs;    -   (ii) thawing the portion of the first population of        cryopreserved TILs;    -   (iii) performing a first expansion by culturing the portion of        the first population of TILs in a cell culture medium comprising        IL-2, OKT-3, and antigen presenting cells (APCs) for an        additional expansion period (sometimes referred to as a reREP        period) of at least 3 days, to produce a second population of        TILs, wherein the portion from the first population of TILs is        compared to the second population of TILs to obtain a ratio of        the number of TILs, wherein the ratio of the number of TILs in        the second population of TILs to the number of TILs in the        portion of the first population of TILs is greater than 5:1;    -   (iv) determining based on the ratio in step (iii) whether the        first population of TILs is suitable for use in therapeutic        administration to a patient;    -   (v) determining the first population of TILs is suitable for use        in therapeutic administration when the ratio of the number of        TILs in the second population of TILs to the number of TILs in        the first population of TILs is determined to be greater than        5:1 in step (iv).

In some embodiments, the ratio of the number of TILs in the secondpopulation of TILs to the number of TILs in the portion of the firstpopulation of TILs is greater than 50:1.

In some embodiments, the method further comprises performing expansionof the entire first population of cryopreserved TILs from step (i)according to the methods as described in any of the embodiments providedherein.

In some embodiments, the method further comprises administering theentire first population of cryopreserved TILs from step (i) to thepatient.

K. Closed Systems for TIL Manufacturing

The present invention provides for the use of closed systems during theTIL culturing process. Such closed systems allow for preventing and/orreducing microbial contamination, allow for the use of fewer flasks, andallow for cost reductions. In some embodiments, the closed system usestwo containers.

Such closed systems are well-known in the art and can be found, forexample, at http://www.fda.gov/cber/guidelines.htm andhttps://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Blood/ucm076779.htm.

As provided on the FDA website, closed systems with sterile methods areknown and well described. See,https://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Blood/ucm076779.htm, as referenced above and provided inpertinent part below.

Introduction

Sterile connecting devices (STCDs) produce sterile welds between twopieces of compatible tubing. This procedure permits sterile connectionof a variety of containers and tube diameters. This guidance describesrecommended practices and procedures for use of these devices. Thisguidance does not address the data or information that a manufacturer ofa sterile connecting device must submit to FDA in order to obtainapproval or clearance for marketing. It is also important to note thatthe use of an approved or cleared sterile connecting device for purposesnot authorized in the labeling may cause the device to be consideredadulterated and misbranded under the Federal Food, Drug and CosmeticAct.

1. FDA Recommendations

Manufacturers of blood products who propose to routinely use anFDA-cleared STCD should incorporate information regarding such use instandard operating procedure (SOP) manuals for each blood product. Theseentries should include record keeping, product tracking, tube weldquality control, lot numbers of software and disposables (includingsource(s) of elements to be added). Quality control procedures shouldinclude a test of the integrity of each weld.

2. Applications of the STCD

The user should be aware that use of the device may create a new productor significantly modify the configuration of a regulated product forwhich safety and efficacy have not been demonstrated. For those “newproducts” subject to licensure, applications, or application supplementsmust be submitted to FDA in addition to submission of a SOP. In general,pooling or mixing that involves cellular components represents a changein the product that requires submission and approval of a licenseapplication or application supplement. Such applications and applicationsupplements should contain data and descriptions of manufacturingprocedures that demonstrate that the “new product” is safe and effectivefor its intended use throughout the proposed dating period.

The following comments are provided as guidance on the more common usesof an FDA cleared or approved STCD:

L. Adding a New or Smaller Needle to a Blood Collection Set

Using the STCD to add a needle prior to the initiation of a procedure(whole blood collection, platelet pheresis or source plasma collection)is not considered to open a functionally closed system. If a needle isadded during a procedure, only an STCD approved to weld liquid-filledtubing should be used. If the test of weld integrity is satisfactory,the use of an STCD is not considered to open a functionally closedsystem.

Platelets, Pheresis prepared in an open system should be labeled with a24 hour outdate and Platelets, Pheresis products prepared in afunctionally closed system should be labeled with a five day outdate(See Revised Guideline for Collection of Platelets, Pheresis, Oct. 7,1988).

The source and specifications of added tubing and needles should beaddressed in the blood center's SOP and records. Using the STCD to addneedles does not represent a major change in manufacturing for whichlicensed establishments need preapproval.

M. Using the STCD to Prepare Components

When the STCD is used to attach additional component preparation bags,records should be properly maintained identifying the source of thetransfer packs and the appropriate verification of blood unit number andABO/Rh. All blood and blood components must be appropriately labeled (21CFR 606.121).

Examples:

-   -   Adding a fourth bag to a whole blood collection triple-pack for        the production of Cryoprecipitated AHF from Fresh Frozen Plasma.    -   Connection of an additive solution to a red blood cell unit.    -   Addition of an in-line filter that has been FDA cleared for use        in manufacturing components.    -   Addition of a third storage container to a platelet pheresis        harness.    -   For the above stated uses, procedures should be developed and        records maintained, but licensees need not have FDA approval in        order to institute the procedures.

1. Using the STCD to Pool Blood Products

Appropriate use of an STCD to pool Platelets prepared from Whole Bloodcollection may obviate potential contamination from the spike and portentries commonly used. Pooling performed immediately before transfusionis an example of such appropriate use. Pooled Platelets should beadministered not more than 4 hours after pooling (See 21 CFR606.122(1)(2)).

However, pooling and subsequent storage may increase the risk comparedto administration of random donor units; if one contaminated unit ispooled with others and stored before administration, the total bacterialinoculum administered may be increased as a result of replication in theadditional volume. Accordingly, the proposed use of an STCD to pool andstore platelets for more than 4 hours should be supported by data whichsatisfactorily addresses whether such pooling is associated withincreased risk.

Such platelet pooling constitutes manufacture of a new product.

Pooling or mixing that involves platelets is considered the manufactureof a new product that requires submission and approval of a licenseapplication or application supplement if the storage period is to exceedfour hours.

2. Using the STCD to Prepare an Aliquot for Pediatric Use and DividedUnits

Pediatric units and divided units for Whole Blood, Red Blood Cells, andFresh Frozen Plasma prepared using an STCD will not be considered a newproduct for which a biologics license application (BLA) supplement isrequired providing the following conditions are met:

-   -   The manufacturer should have an approved biologics license or        license supplement, for the original (i.e., undivided) product,        including approval for each anticoagulant used.    -   Labels should be submitted for review and approval before        distribution. A notation should be made under the comments        section of FDA Form 2567, Transmittal of Labels and Circulars.    -   Final product containers approved for storage of the component        being prepared should be used.

Platelets manufactured under licensure must contain at least 5.5×(10)¹⁰platelets (21 CFR 640.24 (c)). Platelets, Pheresis manufactured underlicensure should contain at least 3.0×(10)¹¹ platelets (See RevisedGuideline for the Collection of Platelets, Pheresis, Oct. 7, 1988).

Procedures to be followed regarding the use of an STCD to preparedivided products from Whole Blood collections and from plasma andplatelets prepared by automated hemapheresis procedures should includedescriptions of:

-   -   How the apheresis harness or collection container will be        modified with an FDA-cleared STCD;    -   the minimum volume of the split plasma or whole blood products;    -   the volume and platelet concentration of the split platelet        pheresis products;    -   storage time of the product. The product should be in an        approved container and should be consistent with the storage        time on the label of such container;    -   method(s) to be used to label and track divided products in the        blood center's records.

NOTE: Procedures for labeling the aliquots should be clearly stated inthe procedure record keeping should be adequate to permit tracking andrecall of all components, if necessary.

3. Using an STCD to Connect Additional Saline or Anticoagulant LinesDuring an Automated Plasmapheresis Procedure

Procedures should be developed and records maintained consistent withthe instrument manufacturer's directions for use, but licensees need nothave FDA approval in order to institute the procedures.

4. Using the STCD to Attach Processing Solutions

When using an STCD to attach containers with processing solutions towashed or frozen red blood cell products, the dating period for theresulting products is 24 hours, unless data are provided in the form oflicense applications or application supplements to CBER to support alonger dating period (21 CFR 610.53(c)). Exemptions or modificationsmust be approved in writing from the Director, CBER (21 CFR 610.53(d)).

5. Using an STCD to Add an FDA-Cleared Leukocyte Reduction Filter

Some leuko-reduction filters are not integrally attached to the WholeBlood collection systems. Procedures for use of an STCD for pre-storagefiltration should be consistent with filter manufacturers' directionsfor use.

Leukocyte reduction prior to issue constitutes a major manufacturingchange. Therefore, for new leukocyte-reduced products prepared using anSTCD, manufacturers must submit biologics license applications (21 CFR601.2) or prior approval application supplements to FDA (21 CFR 601.12).

Using an STCD to remove samples from blood product containers fortesting (e.g., using an STCD to obtain a sample of platelets from acontainer of Platelets or Platelets, Pheresis for cross matching).

If the volume and/or cell count of the product after sample withdrawaldiffer from what is stated on the original label or in the circular ofinformation, the label on the product should be modified to reflect thenew volume and/or cell count. For example, samples may not be removedthat reduce the platelet count of a unit of Platelets to less than5.5×(10)¹⁰ platelets (21 CFR 640.24 (c)).

6. Additional Information from FDA Guidance

The FDA guidance presents general guidance as well as specificinformation and examples concerning specifications for submission ofapplications and application supplements to FDA addressing use of anSTCD. If further questions arise concerning appropriate use of an STCD,concerns should be directed to the Office of Blood Research and Review,Center for Biologics Evaluation and Research.

In some embodiments, the closed system uses one container from the timethe tumor fragments are obtained until the TILs are ready foradministration to the patient or cryopreserving. In some embodimentswhen two containers are used, the first container is a closedG-container and the population of TILs is centrifuged and transferred toan infusion bag without opening the first closed G-container. In someembodiments, when two containers are used, the infusion bag is aHypoThermosol-containing infusion bag. A closed system or closed TILcell culture system is characterized in that once the tumor sampleand/or tumor fragments have been added, the system is tightly sealedfrom the outside to form a closed environment free from the invasion ofbacteria, fungi, and/or any other microbial contamination.

In some embodiments, the reduction in microbial contamination is betweenabout 5% and about 100%. In some embodiments, the reduction in microbialcontamination is between about 5% and about 95%. In some embodiments,the reduction in microbial contamination is between about 5% and about90%. In some embodiments, the reduction in microbial contamination isbetween about 10% and about 90%. In some embodiments, the reduction inmicrobial contamination is between about 15% and about 85%. In someembodiments, the reduction in microbial contamination is about 5%, about10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%,about 99%, or about 100%.

The closed system allows for TIL growth in the absence and/or with asignificant reduction in microbial contamination.

Moreover, pH, carbon dioxide partial pressure and oxygen partialpressure of the TIL cell culture environment each vary as the cells arecultured. Consequently, even though a medium appropriate for cellculture is circulated, the closed environment still needs to beconstantly maintained as an optimal environment for TIL proliferation.To this end, it is desirable that the physical factors of pH, carbondioxide partial pressure and oxygen partial pressure within the cultureliquid of the closed environment be monitored by means of a sensor, thesignal whereof is used to control a gas exchanger installed at the inletof the culture environment, and the that gas partial pressure of theclosed environment be adjusted in real time according to changes in theculture liquid so as to optimize the cell culture environment. In someembodiments, the present invention provides a closed cell culture systemwhich incorporates at the inlet to the closed environment a gasexchanger equipped with a monitoring device which measures the pH,carbon dioxide partial pressure and oxygen partial pressure of theclosed environment, and optimizes the cell culture environment byautomatically adjusting gas concentrations based on signals from themonitoring device.

In some embodiments, the pressure within the closed environment iscontinuously or intermittently controlled. That is, the pressure in theclosed environment can be varied by means of a pressure maintenancedevice for example, thus ensuring that the space is suitable for growthof TILs in a positive pressure state, or promoting exudation of fluid ina negative pressure state and thus promoting cell proliferation. Byapplying negative pressure intermittently, moreover, it is possible touniformly and efficiently replace the circulating liquid in the closedenvironment by means of a temporary shrinkage in the volume of theclosed environment.

In some embodiments, optimal culture components for proliferation of theTILs can be substituted or added, and including factors such as IL-2and/or OKT3, as well as combination, can be added.

C. Cell Cultures

In an embodiment, a method for expanding TILs, including those discussabove as well as exemplified in FIG. 27, may include using about 5,000mL to about 25,000 mL of cell medium, about 5,000 mL to about 10,000 mLof cell medium, or about 5,800 mL to about 8,700 mL of cell medium. Insome embodiments, the media is a serum free medium, as described forexample in Example 21. In some embodiments, the media in the firstexpansion is serum free. In some embodiments, the media in the secondexpansion is serum free. In some embodiments, the media in the firstexpansion and the second are both serum free. In an embodiment,expanding the number of TILs uses no more than one type of cell culturemedium. Any suitable cell culture medium may be used, e.g., AIM-V cellmedium (L-glutamine, 50 μM streptomycin sulfate, and 10 μM gentamicinsulfate) cell culture medium (Invitrogen, Carlsbad Calif.). In thisregard, the inventive methods advantageously reduce the amount of mediumand the number of types of medium required to expand the number of TIL.In an embodiment, expanding the number of TIL may comprise feeding thecells no more frequently than every third or fourth day. Expanding thenumber of cells in a gas permeable container simplifies the proceduresnecessary to expand the number of cells by reducing the feedingfrequency necessary to expand the cells.

In an embodiment, the cell medium in the first and/or second gaspermeable container is unfiltered. The use of unfiltered cell medium maysimplify the procedures necessary to expand the number of cells. In anembodiment, the cell medium in the first and/or second gas permeablecontainer lacks beta-mercaptoethanol (BME).

In an embodiment, the duration of the method comprising obtaining atumor tissue sample from the mammal; culturing the tumor tissue samplein a first gas permeable container containing cell medium therein;obtaining TILs from the tumor tissue sample; expanding the number ofTILs in a second gas permeable container containing cell medium for aduration of about 7 to 14 days, e.g., about 11 days. In some embodimentspre-REP is about 7 to 14 days, e.g., about 11 days. In some embodiments,REP is about 7 to 14 days, e.g., about 11 days.

In an embodiment, TILs are expanded in gas-permeable containers.Gas-permeable containers have been used to expand TILs using PBMCs usingmethods, compositions, and devices known in the art, including thosedescribed in U.S. Patent Application Publication No. 2005/0106717 A1,the disclosures of which are incorporated herein by reference. In anembodiment, TILs are expanded in gas-permeable bags. In an embodiment,TILs are expanded using a cell expansion system that expands TILs in gaspermeable bags, such as the Xuri Cell Expansion System W25 (GEHealthcare). In an embodiment, TILs are expanded using a cell expansionsystem that expands TILs in gas permeable bags, such as the WAVEBioreactor System, also known as the Xuri Cell Expansion System W5 (GEHealthcare). In an embodiment, the cell expansion system includes a gaspermeable cell bag with a volume selected from the group consisting ofabout 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL,about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L,about 9 L, and about 10 L.

In an embodiment, TILs can be expanded in G-Rex flasks (commerciallyavailable from Wilson Wolf Manufacturing). Such embodiments allow forcell populations to expand from about 5×10⁵ cells/cm² to between 10×10⁶and 30×10⁶ cells/cm². In an embodiment this is without feeding. In anembodiment, this is without feeding so long as medium resides at aheight of about 10 cm in the G-Rex flask. In an embodiment this iswithout feeding but with the addition of one or more cytokines. In anembodiment, the cytokine can be added as a bolus without any need to mixthe cytokine with the medium. Such containers, devices, and methods areknown in the art and have been used to expand TILs, and include thosedescribed in U.S. Patent Application Publication No. US 2014/0377739A1,International Publication No. WO 2014/210036 A1, U.S. Patent ApplicationPublication No. us 2013/0115617 A1, International Publication No. WO2013/188427 A1, U.S. Patent Application Publication No. US 2011/0136228A1, U.S. Pat. No. 8,809,050 B2, International publication No. WO2011/072088 A2, U.S. Patent Application Publication No. US 2016/0208216A1, U.S. Patent Application Publication No. US 2012/0244133 A1,International Publication No. WO 2012/129201 A1, U.S. Patent ApplicationPublication No. US 2013/0102075 A1, U.S. Pat. No. 8,956,860 B2,International Publication No. WO 2013/173835 A1, U.S. Patent ApplicationPublication No. US 2015/0175966 A1, the disclosures of which areincorporated herein by reference. Such processes are also described inJin et al., J. Immunotherapy, 2012, 35:283-292.

D. Optional Genetic Engineering of TILs

In some embodiments, the TILs are optionally genetically engineered toinclude additional functionalities, including, but not limited to, ahigh-affinity T cell receptor (TCR), e.g., a TCR targeted at atumor-associated antigen such as MAGE-1, HER2, or NY-ESO-1, or achimeric antigen receptor (CAR) which binds to a tumor-associated cellsurface molecule (e.g., mesothelin) or lineage-restricted cell surfacemolecule (e.g., CD19).

E. Optional Cryopreservation of TILs

Either the bulk TIL population or the expanded population of TILs can beoptionally cryopreserved. In some embodiments, cryopreservation occurson the therapeutic TIL population. In some embodiments, cryopreservationoccurs on the TILs harvested after the second expansion. In someembodiments, cryopreservation occurs on the TILs in exemplary Step F ofFIG. 27. In some embodiments, the TILs are cryopreserved in the infusionbag. In some embodiments, the TILs are cryopreserved prior to placementin an infusion bag. In some embodiments, the TILs are cryopreserved andnot placed in an infusion bag. In some embodiments, cryopreservation isperformed using a cryopreservation medium. In some embodiments, thecryopreservation media contains dimethylsulfoxide (DMSO). This isgenerally accomplished by putting the TIL population into a freezingsolution, e.g. 85% complement inactivated AB serum and 15% dimethylsulfoxide (DMSO). The cells in solution are placed into cryogenic vialsand stored for 24 hours at −80° C., with optional transfer to gaseousnitrogen freezers for cryopreservation. See, Sadeghi, et al., ActaOncologica 2013, 52, 978-986.

When appropriate, the cells are removed from the freezer and thawed in a37° C. water bath until approximately ⅘ of the solution is thawed. Thecells are generally resuspended in complete media and optionally washedone or more times. In some embodiments, the thawed TILs can be countedand assessed for viability as is known in the art.

In a preferred embodiment, a population of TILs is cryopreserved usingCS10 cryopreservation media (CryoStor 10, BioLife Solutions). In apreferred embodiment, a population of TILs is cryopreserved using acryopreservation media containing dimethylsulfoxide (DMSO). In apreferred embodiment, a population of TILs is cryopreserved using a 1:1(vol:vol) ratio of CS10 and cell culture media. In a preferredembodiment, a population of TILs is cryopreserved using about a 1:1(vol:vol) ratio of CS10 and cell culture media, further comprisingadditional IL-2.

As discussed above in Steps A through E, cryopreservation can occur atnumerous points throughout the TIL expansion process. In someembodiments, the bulk TIL population after the first expansion accordingto Step B or the expanded population of TILs after the one or moresecond expansions according to Step D can be cryopreserved.Cryopreservation can be generally accomplished by placing the TILpopulation into a freezing solution, e.g., 85% complement inactivated ABserum and 15% dimethyl sulfoxide (DMSO). The cells in solution areplaced into cryogenic vials and stored for 24 hours at −80° C., withoptional transfer to gaseous nitrogen freezers for cryopreservation. SeeSadeghi, et al., Acta Oncologica 2013, 52, 978-986.

When appropriate, the cells are removed from the freezer and thawed in a37° C. water bath until approximately ⅘ of the solution is thawed. Thecells are generally resuspended in complete media and optionally washedone or more times. In some embodiments, the thawed TILs can be countedand assessed for viability as is known in the art.

In some cases, the Step B TIL population can be cryopreservedimmediately, using the protocols discussed below. Alternatively, thebulk TIL population can be subjected to Step C and Step D and thencryopreserved after Step D. Similarly, in the case where geneticallymodified TILs will be used in therapy, the Step B or Step D TILpopulations can be subjected to genetic modifications for suitabletreatments.

F. Optional Cell Viability Analyses

Optionally, a cell viability assay can be performed after the firstexpansion (sometimes referred to as the initial bulk expansion), usingstandard assays known in the art. For example, a trypan blue exclusionassay can be done on a sample of the bulk TILs, which selectively labelsdead cells and allows a viability assessment. Other assays for use intesting viability can include but are not limited to the Alamar blueassay; and the MTT assay.

1. Cell Counts, Viability, Flow Cytometry

In some embodiments, cell counts and/or viability are measured. Theexpression of markers such as but not limited CD3, CD4, CD8, and CD56,as well as any other disclosed or described herein, can be measured byflow cytometry with antibodies, for example but not limited to thosecommercially available from BD Bio-sciences (BD Biosciences, San Jose,Calif.) using a FACSCanto™ flow cytometer (BD Biosciences). The cellscan be counted manually using a disposable c-chip hemocytometer (VWR,Batavia, Ill.) and viability can be assessed using any method known inthe art, including but not limited to trypan blue staining.

In some cases, the bulk TIL population can be cryopreserved immediately,using the protocols discussed below. Alternatively, the bulk TILpopulation can be subjected to REP and then cryopreserved as discussedbelow. Similarly, in the case where genetically modified TILs will beused in therapy, the bulk or REP TIL populations can be subjected togenetic modifications for suitable treatments.

2. Cell Cultures

In an embodiment, a method for expanding TILs may include using about5,000 mL to about 25,000 mL of cell medium, about 5,000 mL to about10,000 mL of cell medium, or about 5,800 mL to about 8,700 mL of cellmedium. In an embodiment, expanding the number of TILs uses no more thanone type of cell culture medium. Any suitable cell culture medium may beused, e.g., AIM-V cell medium (L-glutamine, 50 μM streptomycin sulfate,and 10 μM gentamicin sulfate) cell culture medium (Invitrogen, CarlsbadCalif.). In this regard, the inventive methods advantageously reduce theamount of medium and the number of types of medium required to expandthe number of TIL. In an embodiment, expanding the number of TIL maycomprise feeding the cells no more frequently than every third or fourthday. Expanding the number of cells in a gas permeable containersimplifies the procedures necessary to expand the number of cells byreducing the feeding frequency necessary to expand the cells.

In an embodiment, the cell medium in the first and/or second gaspermeable container is unfiltered. The use of unfiltered cell medium maysimplify the procedures necessary to expand the number of cells. In anembodiment, the cell medium in the first and/or second gas permeablecontainer lacks beta-mercaptoethanol (BME).

In an embodiment, the duration of the method comprising obtaining atumor tissue sample from the mammal; culturing the tumor tissue samplein a first gas permeable container containing cell medium therein;obtaining TILs from the tumor tissue sample; expanding the number ofTILs in a second gas permeable container containing cell medium thereinusing aAPCs for a duration of about 14 to about 42 days, e.g., about 28days.

In an embodiment, TILs are expanded in gas-permeable containers.Gas-permeable containers have been used to expand TILs using PBMCs usingmethods, compositions, and devices known in the art, including thosedescribed in U.S. Patent Application Publication No. 2005/0106717 A1,the disclosures of which are incorporated herein by reference. In anembodiment, TILs are expanded in gas-permeable bags. In an embodiment,TILs are expanded using a cell expansion system that expands TILs in gaspermeable bags, such as the Xuri Cell Expansion System W25 (GEHealthcare). In an embodiment, TILs are expanded using a cell expansionsystem that expands TILs in gas permeable bags, such as the WAVEBioreactor System, also known as the Xuri Cell Expansion System W5 (GEHealthcare). In an embodiment, the cell expansion system includes a gaspermeable cell bag with a volume selected from the group consisting ofabout 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL,about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L,about 9 L, and about 10 L.

In an embodiment, TILs can be expanded in G-Rex flasks (commerciallyavailable from Wilson Wolf Manufacturing). Such embodiments allow forcell populations to expand from about 5×10⁵ cells/cm² to between 10×10⁶and 30×10⁶ cells/cm². In an embodiment this is without feeding. In anembodiment, this is without feeding so long as medium resides at aheight of about 10 cm in the G-Rex flask. In an embodiment this iswithout feeding but with the addition of one or more cytokines. In anembodiment, the cytokine can be added as a bolus without any need to mixthe cytokine with the medium. Such containers, devices, and methods areknown in the art and have been used to expand TILs, and include thosedescribed in U.S. Patent Application Publication No. US 2014/0377739A1,International Publication No. WO 2014/210036 A1, U.S. Patent ApplicationPublication No. us 2013/0115617 A1, International Publication No. WO2013/188427 A1, U.S. Patent Application Publication No. US 2011/0136228A1, U.S. Pat. No. 8,809,050 B2, International publication No. WO2011/072088 A2, U.S. Patent Application Publication No. US 2016/0208216A1, U.S. Patent Application Publication No. US 2012/0244133 A1,International Publication No. WO 2012/129201 A1, U.S. Patent ApplicationPublication No. US 2013/0102075 A1, U.S. Pat. No. 8,956,860 B2,International Publication No. WO 2013/173835 A1, U.S. Patent ApplicationPublication No. US 2015/0175966 A1, the disclosures of which areincorporated herein by reference. Such processes are also described inJin et al., J. Immunotherapy, 2012, 35:283-292. Optional GeneticEngineering of TILs

In some embodiments, the TILs are optionally genetically engineered toinclude additional functionalities, including, but not limited to, ahigh-affinity T cell receptor (TCR), e.g., a TCR targeted at atumor-associated antigen such as MAGE-1, HER2, or NY-ESO-1, or achimeric antigen receptor (CAR) which binds to a tumor-associated cellsurface molecule (e.g., mesothelin) or lineage-restricted cell surfacemolecule (e.g., CD19).

IV. Methods of Treating Patients

Methods of treatment begin with the initial TIL collection and cultureof TILs. Such methods have been both described in the art by, forexample, Jin et al., J. Immunotherapy, 2012, 35(3):283-292, incorporatedby reference herein in its entirety. Embodiments of methods of treatmentare described throughout the sections below, including the Examples.

The expanded TILs produced according the methods described herein,including for example as described in Steps A through F above oraccording to Steps A through F above (also as shown, for example, inFIG. 27) find particular use in the treatment of patients with cancer(for example, as described in Goff, et al., J. Clinical Oncology, 2016,34(20):2389-239, as well as the supplemental content; incorporated byreference herein in its entirety. In some embodiments, TIL were grownfrom resected deposits of metastatic melanoma as previously described(see, Dudley, et al., J Immunother., 2003, 26:332-342; incorporated byreference herein in its entirety). Fresh tumor can be dissected understerile conditions. A representative sample can be collected for formalpathologic analysis. Single fragments of 2 mm³ to 3 mm³ may be used. Insome embodiments, 5, 10, 15, 20, 25 or 30 samples per patient areobtained. In some embodiments, 20, 25, or 30 samples per patient areobtained. In some embodiments, 20, 22, 24, 26, or 28 samples per patientare obtained. In some embodiments, 24 samples per patient are obtained.Samples can be placed in individual wells of a 24-well plate, maintainedin growth media with high-dose IL-2 (6,000 IU/mL), and monitored fordestruction of tumor and/or proliferation of TIL. Any tumor with viablecells remaining after processing can be enzymatically digested into asingle cell suspension and cryopreserved, as described herein.

In some embodiments, successfully grown TIL can be sampled for phenotypeanalysis (CD3, CD4, CD8, and CD56) and tested against autologous tumorwhen available. TIL can be considered reactive if overnight cocultureyielded interferon-gamma (IFN-γ) levels >200 pg/mL and twice background.(Goff, et al., J Immunother., 2010, 33:840-847; incorporated byreference herein in its entirety). In some embodiments, cultures withevidence of autologous reactivity or sufficient growth patterns can beselected for a second expansion (for example, a second expansion asprovided in according to Step D of FIG. 27), including second expansionsthat are sometimes referred to as rapid expansion (REP). In someembodiments, expanded TILs with high autologous reactivity (for example,high proliferation during a second expansion), are selected for anadditional second expansion. In some embodiments, TILs with highautologous reactivity (for example, high proliferation during secondexpansion as provided in Step D of FIG. 27), are selected for anadditional second expansion according to Step D of FIG. 27.

In some embodiments, the patient is not moved directly to ACT (adoptivecell transfer), for example, in some embodiments, after tumor harvestingand/or a first expansion, the cells are not utilized immediately. Insuch embodiments, TILs can be cryopreserved and thawed 2 days beforeadministration to a patient. In such embodiments, TILs can becryopreserved and thawed 1 day before administration to a patient. Insome embodiments, the TILs can be cryopreserved and thawed immediatelybefore the administration to a patient.

Cell phenotypes of cryopreserved samples of infusion bag TIL can beanalyzed by flow cytometry (e.g., FlowJo) for surface markers CD3, CD4,CD8, CCR7, and CD45RA (BD BioSciences), as well as by any of the methodsdescribed herein. Serum cytokines were measured by using standardenzyme-linked immunosorbent assay techniques. A rise in serum IFN-g wasdefined as >100 pg/mL and greater than 4 3 baseline levels.

In some embodiments, the TILs produced by the methods provided herein,for example those exemplified in FIG. 27, provide for a surprisingimprovement in clinical efficacy of the TILs. In some embodiments, theTILs produced by the methods provided herein, for example thoseexemplified in FIG. 27, exhibit increased clinical efficacy as comparedto TILs produced by methods other than those described herein, includingfor example, methods other than those exemplified in FIG. 27. In someembodiments, the methods other than those described herein includemethods referred to as process 1C and/or Generation 1 (Gen 1). In someembodiments, the increased efficacy is measured by DCR, ORR, and/orother clinical responses. In some embodiments, the TILS produced by themethods provided herein, for example those exemplified in FIG. 27,exhibit a similar time to response and safety profile compared to TILsproduced by methods other than those described herein, including forexample, methods other than those exemplified in FIG. 27, for examplethe Gen 1 process.

In some embodiments, IFN-gamma (IFN-γ) is indicative of treatmentefficacy and/or increased clinical efficacy. In some embodiments, IFN-γin the blood of subjects treated with TILs is indicative of active TILs.In some embodiments, a potency assay for IFN-γ production is employed.IFN-γ production is another measure of cytotoxic potential. IFN-γproduction can be measured by determining the levels of the cytokineIFN-γ in the blood, serum, or TILs ex vivo of a subject treated withTILs prepared by the methods of the present invention, including thoseas described for example in FIG. 27. In some embodiments, an increase inIFN-γ is indicative of treatment efficacy in a patient treated with theTILs produced by the methods of the present invention. In someembodiments, IFN-γ is increased one-fold, two-fold, three-fold,four-fold, or five-fold or more as compared to an untreated patientand/or as compared to a patient treated with TILs prepared using othermethods than those provide herein including for example, methods otherthan those embodied in FIG. 27. In some embodiments, IFN-γ secretion isincreased one-fold as compared to an untreated patient and/or ascompared to a patient treated with TILs prepared using other methodsthan those provide herein including for example, methods other thanthose embodied in FIG. 27. In some embodiments, IFN-γ secretion isincreased two-fold as compared to an untreated patient and/or ascompared to a patient treated with TILs prepared using other methodsthan those provide herein including for example, methods other thanthose embodied in FIG. 27. In some embodiments, IFN-γ secretion isincreased three-fold as compared to an untreated patient and/or ascompared to a patient treated with TILs prepared using other methodsthan those provide herein including for example, methods other thanthose embodied in FIG. 27. In some embodiments, IFN-γ secretion isincreased four-fold as compared to an untreated patient and/or ascompared to a patient treated with TILs prepared using other methodsthan those provide herein including for example, methods other thanthose embodied in FIG. 27. In some embodiments, IFN-γ secretion isincreased five-fold as compared to an untreated patient and/or ascompared to a patient treated with TILs prepared using other methodsthan those provide herein including for example, methods other thanthose embodied in FIG. 27. In some embodiments, IFN-γ is measured usinga Quantikine ELISA kit. In some embodiments, IFN-γ is measured in TILsex vivo of a subject treated with TILs prepared by the methods of thepresent invention, including those as described for example in FIG. 27.In some embodiments, IFN-γ is measured in blood of a subject treatedwith TILs prepared by the methods of the present invention, includingthose as described for example in FIG. 27. In some embodiments, IFN-γ ismeasured in TILs serum of a subject treated with TILs prepared by themethods of the present invention, including those as described forexample in FIG. 27.

In some embodiments, higher average IP-10 is indicative of treatmentefficacy and/or increased clinical efficacy. In some embodiments, higheraverage IP-10 in the blood of subjects treated with TILs is indicativeof active TILs. IP-10 production can be measured by determining thelevels of the IP-10 in the blood of a subject treated with TILs preparedby the methods of the present invention, including those as describedfor example in FIG. 27. In some embodiments, higher average IP-10 isindicative of treatment efficacy in a patient treated with the TILsproduced by the methods of the present invention. In some embodiments,higher average IP-10 correlates to an increase of one-fold, two-fold,three-fold, four-fold, or five-fold or more as compared to an untreatedpatient and/or as compared to a patient treated with TILs prepared usingother methods than those provide herein including for example, methodsother than those embodied in FIG. 27. In some embodiments, higheraverage IP-10 correlates to an increase of one-fold as compared to anuntreated patient and/or as compared to a patient treated with TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 27. In someembodiments, higher average IP-10 correlates to an increase of two-foldas compared to an untreated patient and/or as compared to a patienttreated with TILs prepared using other methods than those provide hereinincluding for example, methods other than those embodied in FIG. 27. Insome embodiments, higher average IP-10 correlates to an increase ofthree-fold as compared to an untreated patient and/or as compared to apatient treated with TILs prepared using other methods than thoseprovide herein including for example, methods other than those embodiedin FIG. 27. In some embodiments, higher average IP-10 correlates to anincrease of four-fold as compared to an untreated patient and/or ascompared to a patient treated with TILs prepared using other methodsthan those provide herein including for example, methods other thanthose embodied in FIG. 27. In some embodiments, higher average IP-10correlates to an increase of five-fold as compared to an untreatedpatient and/or as compared to a patient treated with TILs prepared usingother methods than those provide herein including for example, methodsother than those embodied in FIG. 27. In some embodiments, IP-10 ismeasured in blood of a subject treated with TILs prepared by the methodsof the present invention, including those as described for example inFIG. 27. In some embodiments, IP-10 is measured in TILs serum of asubject treated with TILs prepared by the methods of the presentinvention, including those as described for example in FIG. 27.

In some embodiments, higher average MCP-1 is indicative of treatmentefficacy and/or increased clinical efficacy. In some embodiments, higheraverage MCP-1 in the blood of subjects treated with TILs is indicativeof active TILs. MCP-1 production can be measured by determining thelevels of the MCP-1 in the blood of a subject treated with TILs preparedby the methods of the present invention, including those as describedfor example in FIG. 27. In some embodiments, higher average MCP-1 isindicative of treatment efficacy in a patient treated with the TILsproduced by the methods of the present invention. In some embodiments,higher average MCP-1 correlates to an increase of one-fold, two-fold,three-fold, four-fold, or five-fold or more as compared to an untreatedpatient and/or as compared to a patient treated with TILs prepared usingother methods than those provide herein including for example, methodsother than those embodied in FIG. 27. In some embodiments, higheraverage MCP-1 correlates to an increase of one-fold as compared to anuntreated patient and/or as compared to a patient treated with TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 27. In someembodiments, higher average MCP-1 correlates to an increase of two-foldas compared to an untreated patient and/or as compared to a patienttreated with TILs prepared using other methods than those provide hereinincluding for example, methods other than those embodied in FIG. 27. Insome embodiments, higher average MCP-1 correlates to an increase ofthree-fold as compared to an untreated patient and/or as compared to apatient treated with TILs prepared using other methods than thoseprovide herein including for example, methods other than those embodiedin FIG. 27. In some embodiments, higher average MCP-1 correlates to anincrease of four-fold as compared to an untreated patient and/or ascompared to a patient treated with TILs prepared using other methodsthan those provide herein including for example, methods other thanthose embodied in FIG. 27. In some embodiments, higher average MCP-1correlates to an increase of five-fold as compared to an untreatedpatient and/or as compared to a patient treated with TILs prepared usingother methods than those provide herein including for example, methodsother than those embodied in FIG. 27. In some embodiments, MCP-1 ismeasured in blood of a subject treated with TILs prepared by the methodsof the present invention, including those as described for example inFIG. 27. In some embodiments, MCP-1 is measured in TILs serum of asubject treated with TILs prepared by the methods of the presentinvention, including those as described for example in FIG. 27.

In some embodiments, the TILs prepared by the methods of the presentinvention, including those as described for example in FIG. 27, exhibitincreased polyclonality as compared to TILs produced by other methods,including those not exemplified in FIG. 27, such as for example, methodsreferred to as process 1C methods. In some embodiments, significantlyimproved polyclonality and/or increased polyclonality is indicative oftreatment efficacy and/or increased clinical efficacy. In someembodiments, polyclonality refers to the T-cell repertoire diversity. Insome embodiments, an increase in polyclonality can be indicative oftreatment efficacy with regard to administration of the TILs produced bythe methods of the present invention. In some embodiments, polyclonalityis increased one-fold, two-fold, ten-fold, 100-fold, 500-fold, or1000-fold as compared to TILs prepared using methods than those provideherein including for example, methods other than those embodied in FIG.27. In some embodiments, polyclonality is increased one-fold as comparedto an untreated patient and/or as compared to a patient treated withTILs prepared using other methods than those provide herein includingfor example, methods other than those embodied in FIG. 27. In someembodiments, polyclonality is increased two-fold as compared to anuntreated patient and/or as compared to a patient treated with TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 27. In someembodiments, polyclonality is increased ten-fold as compared to anuntreated patient and/or as compared to a patient treated with TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 27. In someembodiments, polyclonality is increased 100-fold as compared to anuntreated patient and/or as compared to a patient treated with TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 27. In someembodiments, polyclonality is increased 500-fold as compared to anuntreated patient and/or as compared to a patient treated with TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 27. In someembodiments, polyclonality is increased 1000-fold as compared to anuntreated patient and/or as compared to a patient treated with TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 27.

Measures of efficacy can include The disease control rate (DCR)measurements as well as overall response rate (ORR), as known in the artas well as described in the Examples provided herein, including Example28.

1. Methods of Treating Cancers and Other Diseases

The compositions and methods described herein can be used in a methodfor treating diseases. In an embodiment, they are for use in treatinghyperproliferative disorders. They may also be used in treating otherdisorders as described herein and in the following paragraphs.

In some embodiments, the hyperproliferative disorder is cancer. In someembodiments, the hyperproliferative disorder is a solid tumor cancer. Insome embodiments, the solid tumor cancer is selected from the groupconsisting of melanoma, ovarian cancer, cervical cancer, non-small-celllung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancercaused by human papilloma virus, head and neck cancer (including headand neck squamous cell carcinoma (HNSCC)), renal cancer, and renal cellcarcinoma. In some embodiments, the hyperproliferative disorder is ahematological malignancy. In some embodiments, the solid tumor cancer isselected from the group consisting of chronic lymphocytic leukemia,acute lymphoblastic leukemia, diffuse large B cell lymphoma,non-Hodgkin's lymphoma, Hodgkin's lymphoma, follicular lymphoma, andmantle cell lymphoma.

In an embodiment, the invention includes a method of treating a cancerwith a population of TILs, wherein a patient is pre-treated withnon-myeloablative chemotherapy prior to an infusion of TILs according tothe present disclosure. In an embodiment, the non-myeloablativechemotherapy is cyclophosphamide 60 mg/kg/d for 2 days (days 27 and 26prior to TIL infusion) and fludarabine 25 mg/m²/d for 5 days (days 27 to23 prior to TIL infusion). In an embodiment, after non-myeloablativechemotherapy and TIL infusion (at day 0) according to the presentdisclosure, the patient receives an intravenous infusion of IL-2intravenously at 720,000 IU/kg every 8 hours to physiologic tolerance.

Efficacy of the compounds and combinations of compounds described hereinin treating, preventing and/or managing the indicated diseases ordisorders can be tested using various models known in the art, whichprovide guidance for treatment of human disease. For example, models fordetermining efficacy of treatments for ovarian cancer are described,e.g., in Mullany, et al., Endocrinology 2012, 153, 1585-92; and Fong, etal., J. Ovarian Res. 2009, 2, 12. Models for determining efficacy oftreatments for pancreatic cancer are described in Herreros-Villanueva,et al., World J. Gastroenterol. 2012, 18, 1286-1294. Models fordetermining efficacy of treatments for breast cancer are described,e.g., in Fantozzi, Breast Cancer Res. 2006, 8, 212. Models fordetermining efficacy of treatments for melanoma are described, e.g., inDamsky, et al., Pigment Cell & Melanoma Res. 2010, 23, 853-859. Modelsfor determining efficacy of treatments for lung cancer are described,e.g., in Meuwissen, et al., Genes & Development, 2005, 19, 643-664.Models for determining efficacy of treatments for lung cancer aredescribed, e.g., in Kim, Clin. Exp. Otorhinolaryngol. 2009, 2, 55-60;and Sano, Head Neck Oncol. 2009, 1, 32.

In some embodiments, IFN-gamma (IFN-γ) is indicative of treatmentefficacy for hyperproliferative disorder treatment. In some embodiments,IFN-γ in the blood of subjects treated with TILs is indicative of activeTILs. In some embodiments, a potency assay for IFN-γ production isemployed. IFN-γ production is another measure of cytotoxic potential.IFN-γ production can be measured by determining the levels of thecytokine IFN-γ in the blood of a subject treated with TILs prepared bythe methods of the present invention, including those as described forexample in FIG. 27. In some embodiments, the TILs obtained by thepresent method provide for increased IFN-γ in the blood of subjectstreated with the TILs of the present method as compared subjects treatedwith TILs prepared using methods referred to as process 1C, asexemplified in FIG. 83. In some embodiments, an increase in IFN-γ isindicative of treatment efficacy in a patient treated with the TILsproduced by the methods of the present invention. In some embodiments,IFN-γ is increased one-fold, two-fold, three-fold, four-fold, orfive-fold or more as compared to an untreated patient and/or as comparedto a patient treated with TILs prepared using other methods than thoseprovide herein including for example, methods other than those embodiedin FIG. 27. In some embodiments, IFN-γ secretion is increased one-foldas compared to an untreated patient and/or as compared to a patienttreated with TILs prepared using other methods than those provide hereinincluding for example, methods other than those embodied in FIG. 27. Insome embodiments, IFN-γ secretion is increased two-fold as compared toan untreated patient and/or as compared to a patient treated with TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 27. In someembodiments, IFN-γ secretion is increased three-fold as compared to anuntreated patient and/or as compared to a patient treated with TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 27. In someembodiments, IFN-γ secretion is increased four-fold as compared to anuntreated patient and/or as compared to a patient treated with TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 27. In someembodiments, IFN-γ secretion is increased five-fold as compared to anuntreated patient and/or as compared to a patient treated with TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 27. In someembodiments, IFN-γ is measured using a Quantikine ELISA kit. In someembodiments, IFN-γ is measured using a Quantikine ELISA kit. In someembodiments, IFN-γ is measured in TILs ex vivo from a patient treatedwith the TILs produced by the methods of the present invention. In someembodiments, IFN-γ is measured in blood in a patient treated with theTILs produced by the methods of the present invention. In someembodiments, IFN-γ is measured in serum in a patient treated with theTILs produced by the methods of the present invention.

In some embodiments, higher average IP-10 is indicative of treatmentefficacy and/or increased clinical efficacy for hyperproliferativedisorder treatment. In some embodiments, higher average IP-10 in theblood of subjects treated with TILs is indicative of active TILs. Insome embodiments, the TILs obtained by the present method provide forhigher average IP-10 in the blood of subjects treated with the TILs ofthe present method as compared subjects treated with TILs prepared usingmethods referred to as process 1C, as exemplified in FIG. 83. IP-10production can be measured by determining the levels of the IP-10 in theblood of a subject treated with TILs prepared by the methods of thepresent invention, including those as described for example in FIG. 27.In some embodiments, higher average IP-10 is indicative of treatmentefficacy in a patient treated with the TILs produced by the methods ofthe present invention. In some embodiments, higher average IP-10correlates to an increase of one-fold, two-fold, three-fold, four-fold,or five-fold or more as compared to an untreated patient and/or ascompared to a patient treated with TILs prepared using other methodsthan those provide herein including for example, methods other thanthose embodied in FIG. 27. In some embodiments, higher average IP-10correlates to an increase of one-fold as compared to an untreatedpatient and/or as compared to a patient treated with TILs prepared usingother methods than those provide herein including for example, methodsother than those embodied in FIG. 27. In some embodiments, higheraverage IP-10 correlates to an increase of two-fold as compared to anuntreated patient and/or as compared to a patient treated with TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 27. In someembodiments, higher average IP-10 correlates to an increase ofthree-fold as compared to an untreated patient and/or as compared to apatient treated with TILs prepared using other methods than thoseprovide herein including for example, methods other than those embodiedin FIG. 27. In some embodiments, higher average IP-10 correlates to anincrease of four-fold as compared to an untreated patient and/or ascompared to a patient treated with TILs prepared using other methodsthan those provide herein including for example, methods other thanthose embodied in FIG. 27. In some embodiments, higher average IP-10correlates to an increase of five-fold as compared to an untreatedpatient and/or as compared to a patient treated with TILs prepared usingother methods than those provide herein including for example, methodsother than those embodied in FIG. 27.

In some embodiments, higher average MCP-1 is indicative of treatmentefficacy and/or increased clinical efficacy for hyperproliferativedisorder treatment. In some embodiments, higher average MCP-1 in theblood of subjects treated with TILs is indicative of active TILs. Insome embodiments, the TILs obtained by the present method provide forhigher average MCP-1 in the blood of subjects treated with the TILs ofthe present method as compared subjects treated with TILs prepared usingmethods referred to as process 1C, as exemplified in FIG. 83. MCP-1production can be measured by determining the levels of the MCP-1 in theblood of a subject treated with TILs prepared by the methods of thepresent invention, including those as described for example in FIG. 27.In some embodiments, higher average MCP-1 is indicative of treatmentefficacy in a patient treated with the TILs produced by the methods ofthe present invention. In some embodiments, higher average MCP-1correlates to an increase of one-fold, two-fold, three-fold, four-fold,or five-fold or more as compared to an untreated patient and/or ascompared to a patient treated with TILs prepared using other methodsthan those provide herein including for example, methods other thanthose embodied in FIG. 27. In some embodiments, higher average MCP-1correlates to an increase of one-fold as compared to an untreatedpatient and/or as compared to a patient treated with TILs prepared usingother methods than those provide herein including for example, methodsother than those embodied in FIG. 27. In some embodiments, higheraverage MCP-1 correlates to an increase of two-fold as compared to anuntreated patient and/or as compared to a patient treated with TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 27. In someembodiments, higher average MCP-1 correlates to an increase ofthree-fold as compared to an untreated patient and/or as compared to apatient treated with TILs prepared using other methods than thoseprovide herein including for example, methods other than those embodiedin FIG. 27. In some embodiments, higher average MCP-1 correlates to anincrease of four-fold as compared to an untreated patient and/or ascompared to a patient treated with TILs prepared using other methodsthan those provide herein including for example, methods other thanthose embodied in FIG. 27. In some embodiments, higher average MCP-1correlates to an increase of five-fold as compared to an untreatedpatient and/or as compared to a patient treated with TILs prepared usingother methods than those provide herein including for example, methodsother than those embodied in FIG. 27.

In some embodiments, the TILs prepared by the methods of the presentinvention, including those as described for example in FIG. 27, exhibitincreased polyclonality as compared to TILs produced by other methods,including those not exemplified in FIG. 27, such as for example, methodsreferred to as process 1C methods. In some embodiments, significantlyimproved polyclonality and/or increased polyclonality is indicative oftreatment efficacy and/or increased clinical efficacy for cancertreatment. In some embodiments, polyclonality refers to the T-cellrepertoire diversity. In some embodiments, an increase in polyclonalitycan be indicative of treatment efficacy with regard to administration ofthe TILs produced by the methods of the present invention. In someembodiments, polyclonality is increased one-fold, two-fold, ten-fold,100-fold, 500-fold, or 1000-fold as compared to TILs prepared usingmethods than those provide herein including for example, methods otherthan those embodied in FIG. 27. In some embodiments, polyclonality isincreased one-fold as compared to an untreated patient and/or ascompared to a patient treated with TILs prepared using other methodsthan those provide herein including for example, methods other thanthose embodied in FIG. 27. In some embodiments, polyclonality isincreased two-fold as compared to an untreated patient and/or ascompared to a patient treated with TILs prepared using other methodsthan those provide herein including for example, methods other thanthose embodied in FIG. 27. In some embodiments, polyclonality isincreased ten-fold as compared to an untreated patient and/or ascompared to a patient treated with TILs prepared using other methodsthan those provide herein including for example, methods other thanthose embodied in FIG. 27. In some embodiments, polyclonality isincreased 100-fold as compared to an untreated patient and/or ascompared to a patient treated with TILs prepared using other methodsthan those provide herein including for example, methods other thanthose embodied in FIG. 27. In some embodiments, polyclonality isincreased 500-fold as compared to an untreated patient and/or ascompared to a patient treated with TILs prepared using other methodsthan those provide herein including for example, methods other thanthose embodied in FIG. 27. In some embodiments, polyclonality isincreased 1000-fold as compared to an untreated patient and/or ascompared to a patient treated with TILs prepared using other methodsthan those provide herein including for example, methods other thanthose embodied in FIG. 27.

2. Methods of Co-Administration

In some embodiments, the TILs produced as described herein, includingfor example TILs derived from a method described in Steps A through F ofFIG. 27, can be administered in combination with one or more immunecheckpoint regulators, such as the antibodies described below. Forexample, antibodies that target PD-1 and which can be co-administeredwith the TILs of the present invention include, e.g., but are notlimited to nivolumab (BMS-936558, Bristol-Myers Squibb; Opdivo®),pembrolizumab (lambrolizumab, MK03475 or MK-3475, Merck; Keytruda®),humanized anti-PD-1 antibody JS001 (ShangHai JunShi), monoclonalanti-PD-1 antibody TSR-042 (Tesaro, Inc.), Pidilizumab (anti-PD-1 mAbCT-011, Medivation), anti-PD-1 monoclonal Antibody BGB-A317 (BeiGene),and/or anti-PD-1 antibody SHR-1210 (ShangHai HengRui), human monoclonalantibody REGN2810 (Regeneron), human monoclonal antibody MDX-1106(Bristol-Myers Squibb), and/or humanized anti-PD-1 IgG4 antibody PDR001(Novartis). In some embodiments, the PD-1 antibody is from clone:RMP1-14 (rat IgG)—BioXcell cat #BP0146. Other suitable antibodiessuitable for use in co-administration methods with TILs producedaccording to Steps A through F as described herein are anti-PD-1antibodies disclosed in U.S. Pat. No. 8,008,449, herein incorporated byreference. In some embodiments, the antibody or antigen-binding portionthereof binds specifically to PD-L1 and inhibits its interaction withPD-1, thereby increasing immune activity. Any antibodies known in theart which bind to PD-L1 and disrupt the interaction between the PD-1 andPD-L1, and stimulates an anti-tumor immune response, are suitable foruse in co-administration methods with TILs produced according to Steps Athrough F as described herein. For example, antibodies that target PD-L1and are in clinical trials, include BMS-936559 (Bristol-Myers Squibb)and MPDL3280A (Genentech). Other suitable antibodies that target PD-L1are disclosed in U.S. Pat. No. 7,943,743, herein incorporated byreference. It will be understood by one of ordinary skill that anyantibody which binds to PD-1 or PD-L1, disrupts the PD-1/PD-L1interaction, and stimulates an anti-tumor immune response, are suitablefor use in co-administration methods with TILs produced according toSteps A through F as described herein. In some embodiments, the subjectadministered the combination of TILs produced according to Steps Athrough F is co administered with a and anti-PD-1 antibody when thepatient has a cancer type that is refractory to administration of theanti-PD-1 antibody alone. In some embodiments, the patient isadministered TILs in combination with and anti-PD-1 when the patient hasrefractory melanoma. In some embodiments, the patient is administeredTILs in combination with and anti-PD-1 when the patient hasnon-small-cell lung carcinoma (NSCLC).

3. Optional Lymphodepletion Preconditioning of Patients

In an embodiment, the invention includes a method of treating a cancerwith a population of TILs, wherein a patient is pre-treated withnon-myeloablative chemotherapy prior to an infusion of TILs according tothe present disclosure. In an embodiment, the invention includes apopulation of TILs for use in the treatment of cancer in a patient whichhas been pre-treated with non-myeloablative chemotherapy. In anembodiment, the population of TILs is for administration by infusion. Inan embodiment, the non-myeloablative chemotherapy is cyclophosphamide 60mg/kg/d for 2 days (days 27 and 26 prior to TIL infusion) andfludarabine 25 mg/m²/d for 5 days (days 27 to 23 prior to TIL infusion).In an embodiment, after non-myeloablative chemotherapy and TIL infusion(at day 0) according to the present disclosure, the patient receives anintravenous infusion of IL-2 (aldesleukin, commercially available asPROLEUKIN) intravenously at 720,000 IU/kg every 8 hours to physiologictolerance. In certain embodiments, the population of TILs is for use intreating cancer in combination with IL-2, wherein the IL-2 isadministered after the population of TILs.

Experimental findings indicate that lymphodepletion prior to adoptivetransfer of tumor-specific T lymphocytes plays a key role in enhancingtreatment efficacy by eliminating regulatory T cells and competingelements of the immune system (‘cytokine sinks’). Accordingly, someembodiments of the invention utilize a lymphodepletion step (sometimesalso referred to as “immunosuppressive conditioning”) on the patientprior to the introduction of the TILs of the invention.

In general, lymphodepletion is achieved using administration offludarabine or cyclophosphamide (the active form being referred to asmafosfamide) and combinations thereof. Such methods are described inGassner, et al., Cancer Immunol. Immunother. 2011, 60, 75-85, Muranski,et al., Nat. Clin. Pract. Oncol., 2006, 3, 668-681, Dudley, et al., J.Clin. Oncol. 2008, 26, 5233-5239, and Dudley, et al., J. Clin. Oncol.2005, 23, 2346-2357, all of which are incorporated by reference hereinin their entireties.

In some embodiments, the fludarabine is administered at a concentrationof 0.5 μg/mL-10 μg/mL fludarabine. In some embodiments, the fludarabineis administered at a concentration of 1 μg/mL fludarabine. In someembodiments, the fludarabine treatment is administered for 1 day, 2days, 3 days, 4 days, 5 days, 6 days, or 7 days or more. In someembodiments, the fludarabine is administered at a dosage of 10mg/kg/day, 15 mg/kg/day, 20 mg/kg/day, 25 mg/kg/day, 30 mg/kg/day, 35mg/kg/day, 40 mg/kg/day, or 45 mg/kg/day. In some embodiments, thefludarabine treatment is administered for 2-7 days at 35 mg/kg/day. Insome embodiments, the fludarabine treatment is administered for 4-5 daysat 35 mg/kg/day. In some embodiments, the fludarabine treatment isadministered for 4-5 days at 25 mg/kg/day.

In some embodiments, the mafosfamide, the active form ofcyclophosphamide, is obtained at a concentration of 0.5 μg/mL-10 μg/mLby administration of cyclophosphamide. In some embodiments, mafosfamide,the active form of cyclophosphamide, is obtained at a concentration of 1μg/mL by administration of cyclophosphamide. In some embodiments, thecyclophosphamide treatment is administered for 1 day, 2 days, 3 days, 4days, 5 days, 6 days, or 7 days or more. In some embodiments, thecyclophosphamide is administered at a dosage of 100 mg/m²/day, 150mg/m²/day, 175 mg/m²/day 200 mg/m²/day, 225 mg/m²/day, 250 mg/m²/day,275 mg/m²/day, or 300 mg/m²/day. In some embodiments, thecyclophosphamide is administered intravenously (i.e., i.v.) In someembodiments, the cyclophosphamide treatment is administered for 2-7 daysat 35 mg/kg/day. In some embodiments, the cyclophosphamide treatment isadministered for 4-5 days at 250 mg/m²/day i.v. In some embodiments, thecyclophosphamide treatment is administered for 4 days at 250 mg/m²/dayi.v.

In some embodiments, lymphodepletion is performed by administering thefludarabine and the cyclophosphamide together to a patient. In someembodiments, fludarabine is administered at 25 mg/m²/day i.v. andcyclophosphamide is administered at 250 mg/m²/day i.v. over 4 days.

In an embodiment, the lymphodepletion is performed by administration ofcyclophosphamide at a dose of 60 mg/m²/day for two days followed byadministration of fludarabine at a dose of 25 mg/m²/day for five days.

4. IL-2 Regimens

In an embodiment, the IL-2 regimen comprises a high-dose IL-2 regimen,wherein the high-dose IL-2 regimen comprises aldesleukin, or abiosimilar or variant thereof, administered intravenously starting onthe day after administering a therapeutically effective portion of thetherapeutic population of TILs, wherein the aldesleukin or a biosimilaror variant thereof is administered at a dose of 0.037 mg/kg or 0.044mg/kg IU/kg (patient body mass) using 15-minute bolus intravenousinfusions every eight hours until tolerance, for a maximum of 14 doses.Following 9 days of rest, this schedule may be repeated for another 14doses, for a maximum of 28 doses in total.

In an embodiment, the IL-2 regimen comprises a decrescendo IL-2 regimen.Decrescendo IL-2 regimens have been described in O'Day, et al., J. Clin.Oncol. 1999, 17, 2752-61 and Eton, et al., Cancer 2000, 88, 1703-9, thedisclosures of which are incorporated herein by reference. In anembodiment, a decrescendo IL-2 regimen comprises 18×10⁶ IU/m²administered intravenously over 6 hours, followed by 18×10⁶ IU/m²administered intravenously over 12 hours, followed by 18×10⁶ IU/m²administered intravenously over 24 hrs, followed by 4.5×10⁶ IU/m²administered intravenously over 72 hours. This treatment cycle may berepeated every 28 days for a maximum of four cycles. In an embodiment, adecrescendo IL-2 regimen comprises 18,000,000 IU/m² on day 1, 9,000,000IU/m² on day 2, and 4,500,000 IU/m² on days 3 and 4.

In an embodiment, the IL-2 regimen comprises administration of pegylatedIL-2 every 1, 2, 4, 6, 7, 14 or 21 days at a dose of 0.10 mg/day to 50mg/day.

5. Adoptive Cell Transfer

Adoptive cell transfer (ACT) is a very effective form of immunotherapyand involves the transfer of immune cells with antitumor activity intocancer patients. ACT is a treatment approach that involves theidentification, in vitro, of lymphocytes with antitumor activity, the invitro expansion of these cells to large numbers and their infusion intothe cancer-bearing host. Lymphocytes used for adoptive transfer can bederived from the stroma of resected tumors (tumor infiltratinglymphocytes or TILs). TILs for ACT can be prepared as described herein.In some embodiments, the TILs are prepared, for example, according to amethod as described in FIG. 27. They can also be derived or from bloodif they are genetically engineered to express antitumor T-cell receptors(TCRs) or chimeric antigen receptors (CARs), enriched with mixedlymphocyte tumor cell cultures (MLTCs), or cloned using autologousantigen presenting cells and tumor derived peptides. ACT in which thelymphocytes originate from the cancer-bearing host to be infused istermed autologous ACT. U.S. Publication No. 2011/0052530 relates to amethod for performing adoptive cell therapy to promote cancerregression, primarily for treatment of patients suffering frommetastatic melanoma, which is incorporated by reference in its entiretyfor these methods. In some embodiments, TILs can be administered asdescribed herein. In some embodiments, TILs can be administered in asingle dose. Such administration may be by injection, e.g., intravenousinjection. In some embodiments, TILs and/or cytotoxic lymphocytes may beadministered in multiple doses. Dosing may be once, twice, three times,four times, five times, six times, or more than six times per year.Dosing may be once a month, once every two weeks, once a week, or onceevery other day. Administration of TILs and/or cytotoxic lymphocytes maycontinue as long as necessary.

6. Exemplary Treatment Embodiments

In some embodiments, the present disclosure provides a method oftreating a cancer with a population of tumor infiltrating lymphocytes(TILs) comprising the steps of (a) obtaining a first population of TILsfrom a tumor resected from a patient; (b) performing an initialexpansion of the first population of TILs in a first cell culture mediumto obtain a second population of TILs, wherein the second population ofTILs is at least 5-fold greater in number than the first population ofTILs, and wherein the first cell culture medium comprises IL-2; (c)performing a rapid expansion of the second population of TILs using apopulation of myeloid artificial antigen presenting cells (myeloidaAPCs) in a second cell culture medium to obtain a third population ofTILs, wherein the third population of TILs is at least 50-fold greaterin number than the second population of TILs after 7 days from the startof the rapid expansion; and wherein the second cell culture mediumcomprises IL-2 and OKT-3; (d) administering a therapeutically effectiveportion of the third population of TILs to a patient with the cancer. Insome embodiments, the present disclosure a population of tumorinfiltrating lymphocytes (TILs) for use in treating cancer, wherein thepopulation of TILs are obtainable by a method comprising the steps of(b) performing an initial expansion of a first population of TILsobtained from a tumor resected from a patient in a first cell culturemedium to obtain a second population of TILs, wherein the secondpopulation of TILs is at least 5-fold greater in number than the firstpopulation of TILs, and wherein the first cell culture medium comprisesIL-2; (c) performing a rapid expansion of the second population of TILsusing a population of myeloid artificial antigen presenting cells(myeloid aAPCs) in a second cell culture medium to obtain a thirdpopulation of TILs, wherein the third population of TILs is at least50-fold greater in number than the second population of TILs after 7days from the start of the rapid expansion; and wherein the second cellculture medium comprises IL-2 and OKT-3; (d) administering atherapeutically effective portion of the third population of TILs to apatient with the cancer. In some embodiments, the method comprises afirst step (a) of obtaining the first population of TILs from a tumorresected from a patient. In some embodiments, the IL-2 is present at aninitial concentration of about 3000 IU/mL and OKT-3 antibody is presentat an initial concentration of about 30 ng/mL in the second cell culturemedium. In some embodiments, first expansion is performed over a periodnot greater than 14 days. In some embodiments, the first expansion isperformed using a gas permeable container. In some embodiments, thesecond expansion is performed using a gas permeable container. In someembodiments, the ratio of the second population of TILs to thepopulation of aAPCs in the rapid expansion is between 1 to 80 and 1 to400. In some embodiments, the ratio of the second population of TILs tothe population of aAPCs in the rapid expansion is about 1 to 300. Insome embodiments, the cancer for treatment is selected from the groupconsisting of melanoma, ovarian cancer, cervical cancer, non-small-celllung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancercaused by human papilloma virus, head and neck cancer (including headand neck squamous cell carcinoma (HNSCC)), renal cancer, and renal cellcarcinoma. In some embodiments, the cancer for treatment is selectedfrom the group consisting of melanoma, ovarian cancer, and cervicalcancer. In some embodiments, the cancer for treatment is melanoma. Insome embodiments, the cancer for treatment is ovarian cancer. In someembodiments, the cancer for treatment is cervical cancer. In someembodiments, the method of treating cancer further comprises the step oftreating the patient with a non-myeloablative lymphodepletion regimenprior to administering the third population of TILs to the patient. Insome embodiments, the non-myeloablative lymphodepletion regimencomprises the steps of administration of cyclophosphamide at a dose of60 mg/m2/day for two days followed by administration of fludarabine at adose of 25 mg/m2/day for five days. In some embodiments, the high doseIL-2 regimen comprises 600,000 or 720,000 IU/kg of aldesleukin, or abiosimilar or variant thereof, administered as a 15-minute bolusintravenous infusion every eight hours until tolerance.

V. Exemplary Embodiments

In some embodiments, the present invention provides a method fortreating a subject with cancer, the method comprising administeringexpanded tumor infiltrating lymphocytes (TILs) comprising:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a subject by processing a tumor sample obtained from the        patient into multiple tumor fragments;    -   (b) adding the tumor fragments into a closed system;    -   (c) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2 to        produce a second population of TILs, wherein the first expansion        is performed in a closed container providing a first        gas-permeable surface area, wherein the first expansion is        performed for about 11 days to obtain the second population of        TILs, wherein the second population of TILs is at least 50-fold        greater in number than the first population of TILs, and wherein        the transition from step (b) to step (c) occurs without opening        the system;    -   (d) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        third population of TILs, wherein the second expansion is        performed for about 11 days to obtain the third population of        TILs, wherein the third population of TILs is a therapeutic        population of TILs, wherein the second expansion is performed in        a closed container providing a second gas-permeable surface        area, and wherein the transition from step (c) to step (d)        occurs without opening the system;    -   (e) harvesting the therapeutic population of TILs obtained from        step (d), wherein the transition from step (d) to step (e)        occurs without opening the system; and    -   (f) transferring the harvested TIL population from step (e) to        an infusion bag, wherein the transfer from step (e) to (f)        occurs without opening the system;    -   (g) cryopreserving the infusion bag comprising the harvested TIL        population from step (f) using a cryopreservation process; and    -   (h) administering a therapeutically effective dosage of the        third population of TILs from the infusion bag in step (g) to        the patient.

In some embodiment, the cryopreservation process comprisescryopreservation in a media comprising DMSO. In some embodiments, thecryopreservation media comprises 7% to 10% DMSO. In some embodiments,the cryopreservation medium in CS10.

In some embodiments, the therapeutic population of TILs harvested instep (e) comprises sufficient TILs for administering a therapeuticallyeffective dosage of the TILs in step (h).

In some embodiments, the number of TILs sufficient for administering atherapeutically effective dosage in step (h) is from about 2.3×10¹⁰ toabout 13.7×10¹⁰.

In some embodiments, the antigen presenting cells (APCs) are PBMCs.

In some embodiments, the PBMCs are added to the cell culture on any ofdays 9 through 11 in step (d).

In some embodiments, prior to administering a therapeutically effectivedosage of TIL cells in step (h), a non-myeloablative lymphodepletionregimen has been administered to the patient.

In some embodiments, the non-myeloablative lymphodepletion regimencomprises the steps of administration of cyclophosphamide at a dose of60 mg/m²/day for two days followed by administration of fludarabine at adose of 25 mg/m²/day for five days.

In some embodiments, the method further comprises the step of treatingthe patient with a high-dose IL-2 regimen starting on the day afteradministration of the TIL cells to the patient in step (h).

In some embodiments, the high-dose IL-2 regimen comprises 600,000 or720,000 IU/kg administered as a 15-minute bolus intravenous infusionevery eight hours until tolerance.

In some embodiments, the third population of TILs in step (d) providesfor increased efficacy, increased interferon-gamma (IFN-γ) production,increased polyclonality, increased average IP-10, and/or increasedaverage MCP-1 when adminstered to a subject. In some embodiments, theincrease in IFN-γ, increased average IP-10, and/or increased averageMCP-1 is measured in the blood of the subject treated with the TILs.

In some embodiments, the cancer is selected from the group consisting ofmelanoma, ovarian cancer, cervical cancer, non-small-cell lung cancer(NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused byhuman papilloma virus, head and neck cancer (including head and necksquamous cell carcinoma (HNSCC)), renal cancer, and renal cellcarcinoma. In some embodiments, the cancer is selected from the groupconsisting of melanoma, HNSCC, cervical cancers, and NSCLC. In someembodiments, the cancer is melanoma. In some embodiments, the cancer isHNSCC. In some embodiments, the cancer is a cervical cancer. In someembodiments, the cancer is NSCLC.

In an embodiment, the invention provides a method for expanding tumorinfiltrating lymphocytes (TILs).

The present invention provides a method for expanding tumor infiltratinglymphocytes (TILs) comprising: (a) obtaining a tumor sample from apatient, wherein said tumor sample comprises a first population of TILs;(b) processing said tumor sample into multiple tumor fragments; (c)adding said tumor fragments into a closed container; (d) performing aninitial expansion of said first population of TILs in a first cellculture medium to obtain a second population of TILs, wherein said firstcell culture medium comprises IL-2, wherein said initial expansion isperformed in said closed container providing at least 100 cm² ofgas-permeable surface area, wherein said initial expansion is performedwithin a first period of about 7-14 days to obtain a second populationof TILs, wherein said second population of TILs is at least 50-foldgreater in number than said first population of TILs, and wherein thetransition from step (c) to step (d) occurs without opening the system;(e) expanding said second population of TILs in a second cell culturemedium, wherein said second cell culture medium comprises IL-2, OKT-3,and peripheral blood mononuclear cells (PBMCs, also known as mononuclearcells (MNCs)), wherein said expansion is performed within a secondperiod of about 7-14 days to obtain a third population of TILs, whereinsaid third population of TILs exhibits an increased subpopulation ofeffector T cells and/or central memory T cells relative to the secondpopulation of TILs, wherein said expansion is performed in a closedcontainer providing at least 500 cm² of gas-permeable surface area, andwherein the transition from step (d) to step (e) occurs without openingthe system; (f) harvesting said third population of TILs obtained fromstep (e), wherein the transition from step (e) to step (f) occurswithout opening the system; and (g) transferring said harvested TILpopulation from step (f) to an infusion bag, wherein said transfer fromstep (f) to (g) occurs without opening the system. In some embodiments,the method is an in vitro or an ex vivo method.

In some embodiments, the method further comprises harvesting in step (f)via a cell processing system, such as the LOVO system manufactured byFresenius Kabi. The term “LOVO cell processing system” also refers toany instrument or device manufactured by any vendor that can pump asolution comprising cells through a membrane or filter such as aspinning membrane or spinning filter in a sterile and/or closed systemenvironment, allowing for continuous flow and cell processing to removesupernatant or cell culture media without pelletization. In some cases,the cell processing system can perform cell separation, washing,fluid-exchange, concentration, and/or other cell processing steps in aclosed, sterile system.

In some embodiments, the closed container is selected from the groupconsisting of a G-container and a Xuri cellbag.

In some embodiments, the infusion bag in step (g) is aHypoThermosol-containing infusion bag.

In some embodiments, the first period in step (d) and said second periodin step (e) are each individually performed within a period of 10 days,11 days, or 12 days.

In some embodiments, the first period in step (d) and said second periodin step (e) are each individually performed within a period of 11 days.

In some embodiments, steps (a) through (g) are performed within a periodof about 25 days to about 30 days.

In some embodiments, steps (a) through (g) are performed within a periodof about 20 days to about 25 days.

In some embodiments, steps (a) through (g) are performed within a periodof about 20 days to about 22 days.

In some embodiments, steps (a) through (g) are performed in 22 days orless.

In some embodiments, steps (c) through (f) are performed in a singlecontainer, wherein performing steps (c) through (f) in a singlecontainer results in an increase in TIL yield per resected tumor ascompared to performing steps (c) through (f) in more than one container.

In some embodiments, the PBMCs are added to the TILs during the secondperiod in step (e) without opening the system.

In some embodiments, the effector T cells and/or central memory T cellsobtained from said third population of TILs exhibit one or morecharacteristics selected from the group consisting of expressing CD27+,expressing CD28+, longer telomeres, increased CD57 expression, anddecreased CD56 expression relative to effector T cells and/or centralmemory T cells obtained from said second population of cells.

In some embodiments, the effector T cells and/or central memory T cellsobtained from said third population of TILs exhibit increased CD57expression and decreased CD56 expression relative to effector T cellsand/or central memory T cells obtained from said second population ofcells.

In some embodiments, the risk of microbial contamination is reduced ascompared to an open system.

In some embodiments, the TILs from step (g) are infused into a patient.

The present invention also provides a method of treating cancer in apatient with a population of tumor infiltrating lymphocytes (TILs)comprising the steps of: (a) obtaining a tumor sample from a patient,wherein said tumor sample comprises a first population of TILs; (b)processing said tumor sample into multiple tumor fragments; (c) addingsaid tumor fragments into a closed container; (d) performing an initialexpansion of said first population of TILs in a first cell culturemedium to obtain a second population of TILs, wherein said first cellculture medium comprises IL-2, wherein said initial expansion isperformed in said closed container providing at least 100 cm² ofgas-permeable surface area, wherein said initial expansion is performedwithin a first period of about 7-14 days to obtain a second populationof TILs, wherein said second population of TILs is at least 50-foldgreater in number than said first population of TILs, and wherein thetransition from step (c) to step (d) occurs without opening the system;(e) expanding said second population of TILs in a second cell culturemedium, wherein said second cell culture medium comprises IL-2, OKT-3,and peripheral blood mononuclear cells (PBMCs), wherein said expansionis performed within a second period of about 7-14 days to obtain a thirdpopulation of TILs, wherein said third population of TILs exhibits anincreased subpopulation of effector T cells and/or central memory Tcells relative to the second population of TILs, wherein said expansionis performed in a closed container providing at least 500 cm² ofgas-permeable surface area, and wherein the transition from step (d) tostep (e) occurs without opening the system; (f) harvesting said thirdpopulation of TILs obtained from step (e), wherein the transition fromstep (e) to step (f) occurs without opening the system; (g) transferringsaid harvested TIL population from step (f) to an infusion bag, whereinsaid transfer from step (f) to (g) occurs without opening the system;and (h) administering a therapeutically effective amount of TIL cellsfrom said infusion bag in step (g) to said patient.

In some embodiments, the present invention also comprises a populationof tumor infiltrating lymphocytes (TILs) for use in treating cancer,wherein the population of TILs is obtainable from a method comprisingthe steps of: (b) processing a tumor sample obtained from a patientwherein said tumour sample comprises a first population of TILs intomultiple tumor fragments; (c) adding said tumor fragments into a closedcontainer; (d) performing an initial expansion of said first populationof TILs in a first cell culture medium to obtain a second population ofTILs, wherein said first cell culture medium comprises IL-2, whereinsaid initial expansion is performed in said closed container providingat least 100 cm² of gas-permeable surface area, wherein said initialexpansion is performed within a first period of about 7-14 days toobtain a second population of TILs, wherein said second population ofTILs is at least 50-fold greater in number than said first population ofTILs, and wherein the transition from step (c) to step (d) occurswithout opening the system; (e) expanding said second population of TILsin a second cell culture medium, wherein said second cell culture mediumcomprises IL-2, OKT-3, and peripheral blood mononuclear cells (PBMCs),wherein said expansion is performed within a second period of about 7-14days to obtain a third population of TILs, wherein said third populationof TILs exhibits an increased subpopulation of effector T cells and/orcentral memory T cells relative to the second population of TILs,wherein said expansion is performed in a closed container providing atleast 500 cm² of gas-permeable surface area, and wherein the transitionfrom step (d) to step (e) occurs without opening the system; (f)harvesting said third population of TILs obtained from step (e), whereinthe transition from step (e) to step (f) occurs without opening thesystem; (g) transferring said harvested TIL population from step (f) toan infusion bag, wherein said transfer from step (f) to (g) occurswithout opening the system. In some embodiments, the method comprises afirst step (a) obtaining the tumor sample from a patient, wherein saidtumor sample comprises the first population of TILs. In someembodiments, the population of TILs is for administration from saidinfusion bag in step (g) in a therapeutically effective amount.

In some embodiments, prior to administering a therapeutically effectiveamount of TIL cells in step (h), a non-myeloablative lymphodepletionregimen has been administered to said patient. In some embodiments, thepopulations of TILs is for administration to a patient who has undergonea non-myeloablative lymphodepletion regimen.

In some embodiments, the non-myeloablative lymphodepletion regimencomprises the steps of administration of cyclophosphamide at a dose of60 mg/m²/day for two days followed by administration of fludarabine at adose of 25 mg/m²/day for five days.

In some embodiments, the method further comprises the step of treatingsaid patient with a high-dose IL-2 regimen starting on the day afteradministration of said TIL cells to said patient in step (h). In someembodiments, the populations of TILs is for administration prior to ahigh-dose IL-2 regimen. In some embodiments, the population of TILs isfor administration one day before the start of the high-dose IL-2regimen.

In some embodiments, the high-dose IL-2 regimen comprises 600,000 or720,000 IU/kg administered as a 15-minute bolus intravenous infusionevery eight hours until tolerance.

In some embodiments, the effector T cells and/or central memory T cellsobtained from said third population of TILs exhibit one or morecharacteristics selected from the group consisting of expressing CD27+,expressing CD28+, longer telomeres, increased CD57 expression, anddecreased CD56 expression relative to effector T cells and/or centralmemory T cells obtained from said second population of cells.

In some embodiments, the effector T cells and/or central memory T cellsobtained from said third population of TILs exhibit increased CD57expression and decreased CD56 expression relative to effector T cellsand/or central memory T cells obtained from said second population ofcells.

The present invention also provides a method for expanding tumorinfiltrating lymphocytes (TILs) comprising the steps of (a) addingprocessed tumor fragments into a closed system; (b) performing in afirst expansion of said first population of TILs in a first cell culturemedium to obtain a second population of TILs, wherein said first cellculture medium comprises IL-2, wherein said first expansion is performedin a closed container providing a first gas-permeable surface area,wherein said first expansion is performed within a first period of about3-14 days to obtain a second population of TILs, wherein said secondpopulation of TILs is at least 50-fold greater in number than said firstpopulation of TILs, and wherein the transition from step (a) to step (b)occurs without opening the system; (c) expanding said second populationof TILs in a second cell culture medium, wherein said second cellculture medium comprises IL-2, OKT-3, and antigen-presenting cells,wherein said expansion is performed within a second period of about 7-14days to obtain a third population of TILs, wherein said third populationof TILs exhibits an increased subpopulation of effector T cells and/orcentral memory T cells relative to the second population of TILs,wherein said expansion is performed in a closed container providing asecond gas-permeable surface area, and wherein the transition from step(b) to step (c) occurs without opening the system; (d) harvesting saidthird population of TILs obtained from step (c), wherein the transitionfrom step (c) to step (d) occurs without opening the system; and (e)transferring said harvested TIL population from step (d) to an infusionbag, wherein said transfer from step (d) to (e) occurs without openingthe system.

In some embodiments, the method further comprises the step ofcryopreserving the infusion bag comprising the harvested TIL populationusing a cryopreservation process. In some embodiments, thecryopreservation process is performed using a 1:1 ratio of harvested TILpopulation to CS10 media.

In some embodiments, the antigen-presenting cells are peripheral bloodmononuclear cells (PBMCs). In some embodiments, the antigen-presentingcells are artificial antigen-presenting cells.

In some embodiments, the harvesting in step (d) is performed using aLOVO cell processing system.

In some embodiments, the multiple fragments comprise about 50 fragments,wherein each fragment has a volume of about 27 mm³. In some embodiments,the multiple fragments comprise about 30 to about 60 fragments with atotal volume of about 1300 mm³ to about 1500 mm³. In some embodiments,the multiple fragments comprise about 50 fragments with a total volumeof about 1350 mm³. In some embodiments, the multiple fragments compriseabout 50 fragments with a total mass of about 1 gram to about 1.5 grams.

In some embodiments, the second cell culture medium is provided in acontainer selected from the group consisting of a G-container and a Xuricellbag.

In some embodiments, the infusion bag in step (e) is aHypoThermosol-containing infusion bag.

In some embodiments, the first period in step (b) and said second periodin step (c) are each individually performed within a period of 10 days,11 days, or 12 days. In some embodiments, the first period in step (b)and said second period in step (c) are each individually performedwithin a period of 11 days.

In some embodiments, the steps (a) through (e) are performed within aperiod of about 25 days to about 30 days. In some embodiments, the steps(a) through (e) are performed within a period of about 20 days to about25 days. In some embodiments, the steps (a) through (e) are performedwithin a period of about 20 days to about 22 days. In some embodiments,the steps (a) through (e) are performed in 22 days or less. In someembodiments, the steps (a) through (e) and cryopreservation areperformed in 22 days or less.

In some embodiments, the steps (b) through (e) are performed in a singleclosed system, wherein performing steps (b) through (e) in a singlecontainer results in an increase in TIL yield per resected tumor ascompared to performing steps (b) through (e) in more than one container.

In some embodiments, the antigen-presenting cells are added to the TILsduring the second period in step (c) without opening the system.

In some embodiments, the effector T cells and/or central memory T cellsobtained from said third population of TILs exhibit one or morecharacteristics selected from the group consisting of expressing CD27+,expressing CD28+, longer telomeres, increased CD57 expression, anddecreased CD56 expression relative to effector T cells and/or centralmemory T cells obtained from said second population of cells.

In some embodiments, the effector T cells and/or central memory T cellsobtained from said third population of TILs exhibit increased CD57expression and decreased CD56 expression relative to effector T cellsand/or central memory T cells obtained from said second population ofcells.

In some embodiments, the risk of microbial contamination is reduced ascompared to an open system.

In some embodiments, the TILs from step (e) are infused into a patient.

In some embodiments, the closed container comprises a single bioreactor.In some embodiments, the closed container comprises a G-REX-10. In someembodiments, the closed container comprises a G-REX-100. In someembodiments, the closed container comprises a G-Rex 500. In someembodiments, the closed container comprises a Xuri or Wave bioreactorgas permeable bag.

In some embodiments, the present disclosure provides a method forexpanding tumor infiltrating lymphocytes (TILs) into a therapeuticpopulation of TILs comprising:

-   -   (b) adding tumor fragments into a closed system wherein the        tumour fragments comprise a first population of TILs;    -   (c) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2 to        produce a second population of TILs, wherein the first expansion        is performed in a closed container providing a first        gas-permeable surface area, wherein the first expansion is        performed for about 3-14 days to obtain the second population of        TILs, wherein the second population of TILs is at least 50-fold        greater in number than the first population of TILs, and wherein        the transition from step (b) to step (c) occurs without opening        the system;    -   (d) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        third population of TILs, wherein the second expansion is        performed for about 7-14 days to obtain the third population of        TILs, wherein the third population of TILs is a therapeutic        population of TILs which comprises an increased subpopulation of        effector T cells and/or central memory T cells relative to the        second population of TILs, wherein the second expansion is        performed in a closed container providing a second gas-permeable        surface area, and wherein the transition from step (c) to        step (d) occurs without opening the system;    -   (e) harvesting the therapeutic population of TILs obtained from        step (d), wherein the transition from step (d) to step (e)        occurs without opening the system; and    -   (f) transferring the harvested TIL population from step (e) to        an infusion bag, wherein the transfer from step (e) to (f)        occurs without opening the system.

In some embodiments, the method also comprises as a first step:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient by processing a tumor sample obtained from the        patient into multiple tumor fragments.

In an embodiment, the method is an in vitro or an ex vivo method.

In some embodiments, the present disclosure provides a method forexpanding tumor infiltrating lymphocytes (TILs) into a therapeuticpopulation of TILs comprising:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient by processing a tumor sample obtained from the        patient into multiple tumor fragments;    -   (b) adding the tumor fragments into a closed system;    -   (c) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2 to        produce a second population of TILs, wherein the first expansion        is performed in a closed container providing a first        gas-permeable surface area, wherein the first expansion is        performed for about 3-14 days to obtain the second population of        TILs, wherein the second population of TILs is at least 50-fold        greater in number than the first population of TILs, and wherein        the transition from step (b) to step (c) occurs without opening        the system;    -   (d) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        third population of TILs, wherein the second expansion is        performed for about 7-14 days to obtain the third population of        TILs, wherein the third population of TILs is a therapeutic        population of TILs which comprises an increased subpopulation of        effector T cells and/or central memory T cells relative to the        second population of TILs, wherein the second expansion is        performed in a closed container providing a second gas-permeable        surface area, and wherein the transition from step (c) to        step (d) occurs without opening the system;    -   (e) harvesting the therapeutic population of TILs obtained from        step (d), wherein the transition from step (d) to step (e)        occurs without opening the system; and    -   (f) transferring the harvested TIL population from step (e) to        an infusion bag, wherein the transfer from step (e) to (f)        occurs without opening the system.

In an embodiment, the method is an in vitro or an ex vivo method.

In some embodiments, the method further comprises the step ofcryopreserving the infusion bag comprising the harvested TIL populationin step (f) using a cryopreservation process.

In some embodiments, the cryopreservation process is performed using a1:1 ratio of harvested TIL population to cryopreservation media. In someembodiments, the cryopreservation media comprises dimethylsulfoxide. Insome embodiments, the cryopreservation media is selected from the groupconsisting of Cryostor CS10, HypoThermasol, or a combination thereof.

In some embodiments, the antigen-presenting cells are peripheral bloodmononuclear cells (PBMCs).

In some embodiments, the PBMCs are irradiated and allogeneic.

In some embodiments, the PBMCs are added to the cell culture on any ofdays 9 through 14 in step (d).

In some embodiments, the antigen-presenting cells are artificialantigen-presenting cells.

In some embodiments, the harvesting in step (e) is performing using aLOVO cell processing system.

In some embodiments, the tumor fragments are multiple fragments andcomprise about 4 to about 50 fragments, wherein each fragment has avolume of about 27 mm³. In some embodiments, the multiple fragmentscomprise about 30 to about 60 fragments with a total volume of about1300 mm³ to about 1500 mm³. In some embodiments, the multiple fragmentscomprise about 50 fragments with a total volume of about 1350 mm³. Insome embodiments, the multiple fragments comprise about 50 fragmentswith a total mass of about 1 gram to about 1.5 grams.

In some embodiments, the cell culture medium is provided in a containerselected from the group consisting of a G-container and a Xuri cellbag.

In some embodiments, the infusion bag in step (f) is aHypoThermosol-containing infusion bag.

In some embodiments, the first period in step (c) and the second periodin step (e) are each individually performed within a period of 10 days,11 days, or 12 days. In some embodiments, the first period in step (c)and the second period in step (e) are each individually performed withina period of 11 days. In some embodiments, steps (a) through (f) areperformed within a period of about 25 days to about 30 days. In someembodiments, steps (a) through (f) are performed within a period ofabout 20 days to about 25 days. In some embodiments, steps (a) through(f) are performed within a period of about 20 days to about 22 days. Insome embodiments, steps (a) through (f) are performed in 22 days orless. In some embodiments, steps (a) through (f) and cryopreservationare performed in 22 days or less.

In some embodiments, the therapeutic population of TILs harvested instep (e) comprises sufficient TILs for a therapeutically effectivedosage of the TILs. In some embodiments, the number of TILs sufficientfor a therapeutically effective dosage is from about 2.3×10¹⁰ to about13.7×10¹⁰.

In some embodiments, steps (b) through (e) are performed in a singlecontainer, wherein performing steps (b) through (e) in a singlecontainer results in an increase in TIL yield per resected tumor ascompared to performing steps (b) through (e) in more than one container.

In some embodiments, the antigen-presenting cells are added to the TILsduring the second period in step (d) without opening the system.

In some embodiments, the effector T cells and/or central memory T cellsin the therapeutic population of TILs exhibit one or morecharacteristics selected from the group consisting of expressing CD27+,expressing CD28+, longer telomeres, increased CD57 expression, anddecreased CD56 expression relative to effector T cells, and/or centralmemory T cells obtained from the second population of cells.

In some embodiments, the effector T cells and/or central memory T cellsobtained from the third population of TILs exhibit increased CD57expression and decreased CD56 expression relative to effector T cellsand/or central memory T cells obtained from the second population ofcells.

In some embodiments, the risk of microbial contamination is reduced ascompared to an open system.

In some embodiments, the TILs from step (f) are infused into a patient.

In some embodiments, the multiple fragments comprise about 4 fragments.In some embodiments, the 4 fragments are placed into a G-REX-100. Insome embodiments, the 4 fragments are about 0.5 cm in diameter. In someembodiments, the 4 fragments are placed into a G-REX-100. In someembodiments, the 4 fragments are about 0.1 cm, 0.2 cm, 0.3 cm, 0.4 cm,0.5 cm, 0.6 cm, 0.7 cm, 0.8 cm, 0.9 cm, or 1 cm in diameter. In someembodiments, the 4 fragments are about 0.1 cm, 0.2 cm, 0.3 cm, 0.4 cm,0.5 cm, 0.6 cm, 0.7 cm, 0.8 cm, 0.9 cm, or 1 cm in diameter and areplaced into a G-REX-100. In some embodiments, the 4 fragments are about0.1 cm, 0.2 cm, 0.3 cm, 0.4 cm, 0.5 cm, 0.6 cm, 0.7 cm, 0.8 cm, 0.9 cm,or 1 cm in diameter are placed into a container with an equivalentvolume to a G-REX-100. In some embodiments, the 4 fragments are about0.5 cm in diameter and are placed into a G-REX-100. In some embodiments,the 4 fragments are about 0.5 cm in diameter and are placed into acontainer with an equivalent volume to a G-REX-100.

Further details of steps (a), (b), (c), (d), (e) and (f) are providedherein below, including for example but not limited to the embodimentsdescribed under the headings “STEP A: Obtain Patient Tumor Sample”,“STEP B: First Expansion”, “STEP C: First Expansion to Second ExpansionTransition”, “STEP D: Second Expansion”, “STEP E: Harvest TILS and “STEPF: Final Formulation/Transfer to Infusion Bag”.

In some embodiments, the present disclosure provides methods fortreating a subject with cancer, the method comprising administeringexpanded tumor infiltrating lymphocytes (TILs) comprising:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a subject by processing a tumor sample obtained from the        patient into multiple tumor fragments;    -   (b) adding the tumor fragments into a closed system;    -   (c) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2 to        produce a second population of TILs, wherein the first expansion        is performed in a closed container providing a first        gas-permeable surface area, wherein the first expansion is        performed for about 3-14 days to obtain the second population of        TILs, wherein the second population of TILs is at least 50-fold        greater in number than the first population of TILs, and wherein        the transition from step (b) to step (c) occurs without opening        the system;    -   (d) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        third population of TILs, wherein the second expansion is        performed for about 7-14 days to obtain the third population of        TILs, wherein the third population of TILs is a therapeutic        population of TILs which comprises an increased subpopulation of        effector T cells and/or central memory T cells relative to the        second population of TILs, wherein the second expansion is        performed in a closed container providing a second gas-permeable        surface area, and wherein the transition from step (c) to        step (d) occurs without opening the system;    -   (e) harvesting the therapeutic population of TILs obtained from        step (d), wherein the transition from step (d) to step (e)        occurs without opening the system; and    -   (f) transferring the harvested TIL population from step (e) to        an infusion bag, wherein the transfer from step (e) to (f)        occurs without opening the system;    -   (g) optionally cryopreserving the infusion bag comprising the        harvested TIL population from step (f) using a cryopreservation        process; and    -   (h) administering a therapeutically effective dosage of the        third population of TILs from the infusion bag in step (g) to        the patient.

In some embodiments, the invention provides a therapeutic population oftumor infiltrating lymphocytes (TILs) for use in treating cancer,wherein the population is obtainable from a method comprising the stepsof:

-   -   (b) adding tumor fragments into a closed system wherein the        tumour fragments comprise a first population of TILs;    -   (c) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2 to        produce a second population of TILs, wherein the first expansion        is performed in a closed container providing a first        gas-permeable surface area, wherein the first expansion is        performed for about 3-14 days to obtain the second population of        TILs, wherein the second population of TILs is at least 50-fold        greater in number than the first population of TILs, and wherein        the transition from step (b) to step (c) occurs without opening        the system;    -   (d) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        third population of TILs, wherein the second expansion is        performed for about 7-14 days to obtain the third population of        TILs, wherein the third population of TILs is a therapeutic        population of TILs which comprises an increased subpopulation of        effector T cells and/or central memory T cells relative to the        second population of TILs, wherein the second expansion is        performed in a closed container providing a second gas-permeable        surface area, and wherein the transition from step (c) to        step (d) occurs without opening the system;    -   (e) harvesting the therapeutic population of TILs obtained from        step (d), wherein the transition from step (d) to step (e)        occurs without opening the system; and    -   (f) transferring the harvested TIL population from step (e) to        an infusion bag, wherein the transfer from step (e) to (f)        occurs without opening the system; and    -   (g) optionally cryopreserving the infusion bag comprising the        harvested TIL population from step (f) using a cryopreservation        process.

In some embodiments, the population is obtainable by a method alsocomprising as a first step:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient by processing a tumor sample obtained from the        patient into multiple tumor fragments.

In an embodiment, the method is an in vitro or an ex vivo method.

In some embodiments, any of steps (a) to (f) comprise one or morefeatures disclosed herein, e.g. one or more features disclosed under theheadings “STEP A: Obtain Patient Tumor Sample”, “STEP B: FirstExpansion”, “STEP C: First Expansion to Second Expansion Transition”,“STEP D: Second Expansion”, “STEP E: Harvest TILs and “STEP F: FinalFormulation/Transfer to Infusion Bag”.

In some embodiments, step (g) comprises one or more features disclosedherein, e.g. one or more features disclosed under the heading “STEP H:Optional Cryopreservation of TILs”. In some embodiments, step (h)comprise one or more features disclosed herein, e.g. one or morefeatures disclosed under the heading “STEP F:1 PharmaceuticalCompositions, Dosages and Dosing Regimens”.

In some embodiments, the therapeutic population of TILs harvested instep (e) comprises sufficient TILs for administering a therapeuticallyeffective dosage of the TILs in step (h).

In some embodiments, the number of TILs sufficient for administering atherapeutically effective dosage in step (h) is from about 2.3×10¹⁰ toabout 13.7×10¹⁰.

In some embodiments, the antigen presenting cells (APCs) are PBMCs.

In some embodiments, the PBMCs are added to the cell culture on any ofdays 9 through 14 in step (d).

In some embodiments, prior to administering a therapeutically effectivedosage of TIL cells in step (h), a non-myeloablative lymphodepletionregimen has been administered to the patient.

In some embodiments, there is provided a therapeutic population of tumorinfiltrating lymphocytes (TILs) for use in treating cancer and incombination with a non-myeloablative lymphodepletion regimen. In someembodiments, the non-myeloablative lymphodepletion regimen isadministered prior to administering the therapeutic population of tumorinfiltrating lymphocytes (TILs).

In some embodiments, the non-myeloablative lymphodepletion regimencomprises the steps of administration of cyclophosphamide at a dose of60 mg/m²/day for two days followed by administration of fludarabine at adose of 25 mg/m²/day for five days.

In some embodiments, the step of treating the patient with a high-doseIL-2 regimen starting on the day after administration of the TIL cellsto the patient in step (h).

In some embodiments, there is provided a therapeutic population of tumorinfiltrating lymphocytes (TILs) for use in treating cancer and incombination with high-dose IL-2 regimen. In some embodiments, thehigh-dose IL-2 regimen starts on the day after administration of thetherapeutic population of TIL cells.

In some embodiments, the high-dose IL-2 regimen comprises 600,000 or720,000 IU/kg administered as a 15-minute bolus intravenous infusionevery eight hours until tolerance.

In some embodiments, the effector T cells and/or central memory T cellsin the therapeutic population of TILs exhibit one or morecharacteristics selected from the group consisting of expressing CD27+,expressing CD28+, longer telomeres, increased CD57 expression, anddecreased CD56 expression relative to effector T cells, and/or centralmemory T cells obtained from the second population of cells.

In some embodiments, the effector T cells and/or central memory T cellsin the therapeutic population of TILs exhibit increased CD57 expressionand decreased CD56 expression relative to effector T cells and/orcentral memory T cells obtained from the second population of cells.

The present disclosure also provides methods for expanding tumorinfiltrating lymphocytes (TILs) into a therapeutic population of TILscomprising:

-   -   (a) adding processed tumor fragments from a tumor resected from        a patient into a closed system to obtain a first population of        TILs;    -   (b) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2 to        produce a second population of TILs, wherein the first expansion        is performed in a closed container providing a first        gas-permeable surface area, wherein the first expansion is        performed for about 3-14 days to obtain the second population of        TILs, wherein the second population of TILs is at least 50-fold        greater in number than the first population of TILs, and wherein        the transition from step (a) to step (b) occurs without opening        the system;    -   (c) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        third population of TILs, wherein the second expansion is        performed for about 7-14 days to obtain the third population of        TILs, wherein the third population of TILs is a therapeutic        population of TILs which comprises an increased subpopulation of        effector T cells and/or central memory T cells relative to the        second population of TILs, wherein the second expansion is        performed in a closed container providing a second gas-permeable        surface area, and wherein the transition from step (b) to        step (c) occurs without opening the system;    -   (d) harvesting the therapeutic population of TILs obtained from        step (c), wherein the transition from step (c) to step (d)        occurs without opening the system; and    -   (e) transferring the harvested TIL population from step (d) to        an infusion bag, wherein the transfer from step (d) to (e)        occurs without opening the system.

In some embodiments, the therapeutic population of TILs harvested instep (d) comprises sufficient TILs for a therapeutically effectivedosage of the TILs.

In some embodiments, the number of TILs sufficient for a therapeuticallyeffective dosage is from about 2.3×10¹⁰ to about 13.7×10¹⁰.

In some embodiments, the method further comprises the step ofcryopreserving the infusion bag comprising the harvested TIL populationusing a cryopreservation process.

In some embodiments, the cryopreservation process is performed using a1:1 ratio of harvested TIL population to CS10 media.

In some embodiments, the present disclosure provides methods fortreating a subject with cancer, the method comprising administeringexpanded tumor infiltrating lymphocytes (TILs) comprising:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a subject by processing a tumor sample obtained from the        patient into multiple tumor fragments;    -   (b) adding the tumor fragments into a closed system;    -   (c) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2 to        produce a second population of TILs, wherein the first expansion        is performed in a closed container providing a first        gas-permeable surface area, wherein the first expansion is        performed for about 3-14 days to obtain the second population of        TILs, wherein the second population of TILs is at least 50-fold        greater in number than the first population of TILs, and wherein        the transition from step (b) to step (c) occurs without opening        the system;    -   (d) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        third population of TILs, wherein the second expansion is        performed for about 7-14 days to obtain the third population of        TILs, wherein the third population of TILs is a therapeutic        population of TILs which comprises an increased subpopulation of        effector T cells and/or central memory T cells relative to the        second population of TILs, wherein the second expansion is        performed in a closed container providing a second gas-permeable        surface area, and wherein the transition from step (c) to        step (d) occurs without opening the system;    -   (e) harvesting the therapeutic population of TILs obtained from        step (d), wherein the transition from step (d) to step (e)        occurs without opening the system; and    -   (f) transferring the harvested TIL population from step (e) to        an infusion bag, wherein the transfer from step (e) to (f)        occurs without opening the system;    -   (g) optionally cryopreserving the infusion bag comprising the        harvested TIL population from step (f) using a cryopreservation        process; and    -   (h) administering a therapeutically effective dosage of the        third population of TILs from the infusion bag in step (g) to        the patient.        wherein no selection of TIL population is performed during any        of steps (a) to (h). In an embodiment, no selection of the        second population of TILs (the pre-REP population) based on        phenotype is performed prior to performing the second expansion        of step (d). In an embodiment, no selection of the first        population of TILs, second population of TILs, third population        of TILs, or harvested TIL population based on CD8 expression is        performed during any of steps (a) to (h).

In some embodiments, the present disclosure provides methods fortreating a subject with cancer, the method comprising administeringexpanded tumor infiltrating lymphocytes (TILs) comprising:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a subject by processing a tumor sample obtained from the        patient into multiple tumor fragments;    -   (b) adding the tumor fragments into a closed system;    -   (c) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2 to        produce a second population of TILs, wherein the first expansion        is performed in a closed container providing a first        gas-permeable surface area, wherein the first expansion is        performed for about 3-14 days to obtain the second population of        TILs, wherein the second population of TILs is at least 50-fold        greater in number than the first population of TILs, and wherein        the transition from step (b) to step (c) occurs without opening        the system;    -   (d) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        third population of TILs, wherein the second expansion is        performed for about 7-14 days to obtain the third population of        TILs, wherein the third population of TILs is a therapeutic        population of TILs which comprises an increased subpopulation of        effector T cells and/or central memory T cells relative to the        second population of TILs, wherein the second expansion is        performed in a closed container providing a second gas-permeable        surface area, and wherein the transition from step (c) to        step (d) occurs without opening the system;    -   (e) harvesting the therapeutic population of TILs obtained from        step (d), wherein the transition from step (d) to step (e)        occurs without opening the system; and    -   (f) transferring the harvested TIL population from step (e) to        an infusion bag, wherein the transfer from step (e) to (f)        occurs without opening the system;    -   (g) cryopreserving the infusion bag comprising the harvested TIL        population from step (f) using a cryopreservation process,        wherein the cryopreservation process comprises mixing of a        cryopreservation media with the harvested TIL population;    -   (h) administering a therapeutically effective dosage of the        third population of TILs from the infusion bag in step (g) to        the patient.        wherein no selection of TIL population is performed during any        of steps (a) to (h). In an embodiment, no selection of the        second population of TILs (for example, the pre-REP population)        based on phenotype is performed prior to performing the second        expansion of step (d). In an embodiment, no selection of the        first population of TILs, second population of TILs, third        population of TILs, or harvested TIL population based on CD8        expression is performed during any of steps (a) to (h). In some        embodiments, the non-myeloablative lymphodepletion regimen is        administered prior to administering the harvested TIL        population. In some embodiments, the non-myeloablative        lymphodepletion regimen comprises the steps of administration of        cyclophosphamide at a dose of 60 mg/m²/day for two days followed        by administration of fludarabine at a dose of 25 mg/m²/day for        five days.

In some embodiments, the antigen-presenting cells are peripheral bloodmononuclear cells (PBMCs). In some embodiments, the PBMCs are irradiatedand allogeneic. In some embodiments, the PBMCs are added to the cellculture on any of days 9 through 14 in step (c).

In some embodiments, the antigen-presenting cells are artificialantigen-presenting cells.

In some embodiments, the harvesting in step (d) is performed using aLOVO cell processing system.

In some embodiments, the method comprises harvesting in step (d) is viaa LOVO cell processing system, such as the LOVO system manufactured byFresenius Kabi. The term “LOVO cell processing system” also refers toany instrument or device that can pump a solution comprising cellsthrough a membrane or filter such as a spinning membrane or spinningfilter in a sterile and/or closed system environment, allowing forcontinuous flow and cell processing to remove supernatant or cellculture media without pelletization. In some cases, the cell processingsystem can perform cell separation, washing, fluid-exchange,concentration, and/or other cell processing steps in a closed, sterilesystem.

In some embodiments, the tumor fragments are multiple fragments andcomprise about 4 to about 50 fragments, wherein each fragment has avolume of about 27 mm³. In some embodiments, the multiple fragmentscomprise about 30 to about 60 fragments with a total volume of about1300 mm³ to about 1500 mm³. In some embodiments, the multiple fragmentscomprise about 50 fragments with a total volume of about 1350 mm³. Insome embodiments, the multiple fragments comprise about 50 fragmentswith a total mass of about 1 gram to about 1.5 grams.

In some embodiments, the multiple fragments comprise about 4 fragments.In some embodiments, the 4 fragments are placed into a G-REX-100. Insome embodiments, the 4 fragments are about 0.1 cm, 0.2 cm, 0.3 cm, 0.4cm, 0.5 cm, 0.6 cm, 0.7 cm, 0.8 cm, 0.9 cm, or 1 cm in diameter. In someembodiments, the 4 fragments are about 0.1 cm, 0.2 cm, 0.3 cm, 0.4 cm,0.5 cm, 0.6 cm, 0.7 cm, 0.8 cm, 0.9 cm, or 1 cm in diameter and areplaced into a G-REX-100. In some embodiments, the 4 fragments are about0.1 cm, 0.2 cm, 0.3 cm, 0.4 cm, 0.5 cm, 0.6 cm, 0.7 cm, 0.8 cm, 0.9 cm,or 1 cm in diameter are placed into a container with an equivalentvolume to a G-REX-100. In some embodiments, the 4 fragments are about0.5 cm in diameter and are placed into a G-REX-100. In some embodiments,the 4 fragments are about 0.5 cm in diameter and are placed into acontainer with an equivalent volume to a G-REX-100.

In some embodiments, the cell culture medium is provided in a containerselected from the group consisting of a G-container and a Xuri cellbag.

In some embodiments, the infusion bag in step (e) is aHypoThermosol-containing infusion bag.

In some embodiments, the first period in step (b) and the second periodin step (c) are each individually performed within a period of 10 days,11 days, or 12 days. In some embodiments, the first period in step (b)and the second period in step (c) are each individually performed withina period of 11 days. In some embodiments, steps (a) through (e) areperformed within a period of about 25 days to about 30 days. In someembodiments, steps (a) through (e) are performed within a period ofabout 20 days to about 25 days. In some embodiments, steps (a) through(e) are performed within a period of about 20 days to about 22 days. Insome embodiments, steps (a) through (e) are performed in 22 days orless. In some embodiments, steps (a) through (e) and cryopreservationare performed in 22 days or less.

In some embodiments, steps (b) through (e) are performed in a singlecontainer, wherein performing steps (b) through (e) in a singlecontainer results in an increase in TIL yield per resected tumor ascompared to performing steps (b) through (e) in more than one container.

In some embodiments, the antigen-presenting cells are added to the TILsduring the second period in step (c) without opening the system.

In some embodiments, the effector T cells and/or central memory T cellsobtained in the therapeutic population of TILs exhibit one or morecharacteristics selected from the group consisting of expressing CD27+,expressing CD28+, longer telomeres, increased CD57 expression, anddecreased CD56 expression relative to effector T cells, and/or centralmemory T cells obtained from the second population of cells.

In some embodiments, the effector T cells and/or central memory T cellsobtained in the therapeutic population of TILs exhibit increased CD57expression and decreased CD56 expression relative to effector T cells,and/or central memory T cells obtained from the second population ofcells.

In some embodiments, the risk of microbial contamination is reduced ascompared to an open system.

In some embodiments, the TILs from step (e) are infused into a patient.

In some embodiments, the closed container comprises a single bioreactor.In some embodiments, the closed container comprises a G-REX-10. In someembodiments, the closed container comprises a G-REX-100.

EXAMPLES

The embodiments encompassed herein are now described with reference tothe following examples. These examples are provided for the purpose ofillustration only and the disclosure encompassed herein should in no waybe construed as being limited to these examples, but rather should beconstrued to encompass any and all variations which become evident as aresult of the teachings provided herein.

Example 1: Closed System Assays

As discussed herein, protocols and assays were developed for generatingTIL from patient tumors in a closed system.

This Example describes a novel abbreviated procedure for generatingclinically relevant numbers of TILs from patients' resected tumor tissuein G-REX devices and cryopreservation of the final cell product.Additional aspects of this procedure are described in Examples 2 to 8.

Definitions/Abbreviations

-   BSC—Biological Safety Cabinet-   ° C.—degrees Celsius-   CO₂—Carbon dioxide-   CD3—Cluster of Differentiation 3-   CM1—Complete Medium 1-   CM2—Complete Medium 2-   TIWB—Tumor Isolation Wash Buffer-   CM4—Complete Medium 4-   CRF—Control Rate Freezer-   EtOH—ethanol-   GMP—Good Manufacturing Practice-   IL-2, rIL-2—Interleukin-2, Recombinant human Interleukin-2,-   IU—International Unit-   L—Liter-   LN2—liquid nitrogen-   mL—milliliter-   μl—microliter-   mM—millimolar-   micrometer-   NA—Not Applicable-   PBMC—Peripheral Blood Mononuclear Cell-   PPE—Personal Protective Equipment-   Pre—REP—Initial TIL cultures originating from tumor fragments-   REP—Rapid Expansion Protocol-   TIL—Tumor Infiltrating Lymphocytes-   TIWB—TIL Isolation Wash Buffer-   SOP—Standard Operating Procedure    Procedure    -   1. Advanced preparation: Day 0 (Performed up to 36 hours in        advance)        -   1.1 Prepared TIL Isolation Wash Buffer (TIWB) by            supplementing 500 mL Hanks Balanced Salt Solution with 50            μg/mL Gentamicin. For 10 mg/mL Gentamicin stock solution            transferred 2.5 mL to HBSS. For 50 mg/mL stock solution            transferred 0.5 mL to HBSS.        -   1.2. Prepared CM1 media with GlutaMax™ per LAB-005            “Preparation of media for PreREP and REP” for CM2            instructions”. Store at 4° C. up to 24 hours. Allowed to            warm at 37° C. for at least 1 hour prior to use.        -   1.3. Removed IL-2 aliquot(s) from −20° C. freezer and placed            aliquot(s) in 2-8° C. refrigerator.    -   2. Receipt of tumor tissue        -   2.1. Kept all paperwork received with tumor tissue and            obtained photos of transport container and tumor tissue.        -   2.2. If TempTale was provided printed and saved the            associated document; saved the PDF.        -   2.3. Removed tumor specimen and secondary container (zip top            bag) from shipper and stored at 4° C. until ready for            processing.        -   2.4 Shipped unused tumor either in HypoThermasol or as            frozen fragments in CryoStor CS10 (both commercially            available from BioLife Solutions, Inc.).    -   3. Tumor processing for TIL        -   3.1. Aseptically transferred the following materials to the            BSC, as needed, and labeled according to Table 3 below.

TABLE 3 Materials for tumor isolation. Minimum Item Quantity In-ProcessLabel Tumor 1 N/A Petri dish, 150 mm 1 Dissection Petri dish, 100 mm 4Wash 1, 2, 3, 4 Petri dish, 100 mm 1 Unfavorable Tissue 6 well plate 2Lid Label - “Tumor Fragments” Plate Bottom - “Favorable Tissue” Ruler 2N/A Wash Buffer 1 N/A Forceps 1 N/A Long forceps 1 N/A Scalpel As neededN/A

-   -   -   3.2. Labeled the circles of the Tumor Fragments Dishes with            the letters A-J.        -   3.3. Labeled the undersides of the wells of the Favorable            Tissue Dishes with the letters A-J.        -   3.4. Transferred 5 mL Gentamicin to the HBSS bottle. Labeled            as TIWB.        -   3.5. Swirled to mix.        -   3.6. Pipetted 50 mL TIWB to each of the following:            -   1. Wash 1 dish            -   2. Wash 2 dish            -   3. Wash 3 dish            -   4. Wash 4 dish        -   3.7. Pipetted 2 mL TIWB into wells A-J of the Favorable            Tissue Dish.        -   3.8. Covered the Favorable Tissue Dishes (6-well plate            bottom) with the corresponding Tumor Fragments Dish (6-well            plate lid).        -   3.9. Using long forceps, removed the tumor(s) from the            Specimen bottle and transferred to the Wash 1 dish.        -   3.10. Incubated the tumor at ambient temperature in the Wash            1 dish for 3 minutes.        -   3.11. During the incubation, relabeled the Specimen bottle            “Bioburden” and stored at 2-8° C. until submitted to Quality            Control for testing.        -   3.12. Discarded long forceps and used short forceps for            further manipulations.        -   3.13. Using forceps transferred the tumor to the Wash 2            dish.        -   3.14. Incubated the tumor at ambient temperature in the Wash            2 dish for 3 minutes.        -   3.15. Using forceps transferred the tumor to the Wash 3            dish.        -   3.16. Incubated the tumor at ambient in the Wash 3 dish for            3 minutes.        -   3.17. Removed the Tumor Fragment Dishes (6-well plate lids)            from the Favorable Tissue Dishes (6-well plate bottoms) and            placed the Tumor Fragments Dishes upside down on the BSC            surface.        -   3.18. Using a transfer pipette, added approximately 4            evenly-spaced, individual drops of TIWB to each circle of            the Tumor Fragments dishes.        -   3.19. Placed a ruler underneath the Dissection dish.        -   3.20. Using forceps transferred the tumor to the Dissection            dish.        -   3.21. Using the ruler under the Dissection dish, measured            and recorded the length of the tumor.        -   3.22. For tumors greater than 1 cm additional Favorable            Tissue Dishes were made.        -   3.23. Performed an initial dissection of the tumor pieces in            the Dissection dish into 10 intermediate pieces and care was            taken to conserve the tumor structure of each intermediate            piece.        -   3.24. Transferred any intermediate tumor pieces not being            actively dissected into fragments to the Wash 4 dish to            ensure the tissue remained hydrated during the entire            dissection procedure.        -   3.25. Working with one intermediate tumor piece at a time,            carefully sliced the tumor into up to 3×3×3 mm fragments in            the Dissection Dish, using the ruler underneath the dish for            reference. When scalpel became dull, replaced with a new            scalpel.        -   3.26. Continued dissecting fragments from the intermediate            tumor piece until all tissue in the intermediate piece had            been evaluated.        -   3.27. Selected favorable fragments and using a transfer            pipette transferred up to 4 favorable fragments into the            TIWB drops in one circle in the Tumor Fragments dish.        -   3.28. Using a transfer pipette transferred any remaining            favorable fragments from the tumor piece, when available, to            the corresponding well in the Favorable Tissue Dish.        -   3.29. Using a transfer pipette transferred as much as            possible of the unfavorable tissue and waste product to the            Unfavorable Tissue dish to clear the dissection dish.            Unfavorable tissue was indicated by yellow adipose tissue or            necrotic tissue.        -   3.30. Continued processing by repeating step 7.3.25-7.3.30            for the remaining intermediate tumor pieces, working one            intermediate piece at a time until all of the tumor had been            processed.        -   3.31. If fewer than 4 tumor fragments were available in the            corresponding circle of the Tumor Fragments Dish, it was            acceptable to use fragments from a non-corresponding well of            the Favorable Tissue Dish as available to achieve the 40            fragment goal. When less than 40 fragments, 10-40 were            placed in a singled G-Rex 100M flask.

    -   4. Seeding G-Rex 100M flask        -   4.1. Aseptically transferred the following materials to the            BSC, as needed, and labeled according to the Table 4 below.

TABLE 4 Additional Materials for Seeding Flasks. Minimum Item QuantityIn-Process Label G-Rex 100M flask As Needed Lot# Warm CM1 As Needed Lot#IL-2 Aliquots As Needed Lot#

-   -   -   4.2. Supplemented each liter of CM1 with 1 mL of IL-2 stock            solution (6×10⁶ IU/mL).        -   4.3. Placed 1000 mL of pre-warmed CM1 containing 6,000 IU/mL            of IL-2 in each G-REX 100M bioreactor needed as determined            by Table 5 below.        -   4.4. Using a transfer pipette, transferred the appropriate            number of tumor fragments to each G-Rex 100M flask,            distributing fragments per Table 5.        -   4.5. When one or more tumor fragments transferred to the            G-Rex 100M flask float, obtained one additional tumor            fragment if available from the Favorable Tissue Dish and            transferred it to the G-Rex 100M flask.        -   4.6. Recorded the total number of fragments added to each            flask.        -   4.7. Discarded the Unfavorable Tissue dish.        -   4.8. Placed each G-REX 100M bioreactor in 37° C., 5% CO₂            incubator.        -   4.9. When more than 40 fragments were available:            -   4.9.1. When >41 fragments were obtained, placed 1000 mL                of pre-warmed complete CM1 in a second G-REX 100M                bioreactor.

TABLE 5 Number of G-REX bioreactors needed. Number of Number ofFragments G-REX G-REX CM1 needed 1-40 G-REX 100M 1 1000 mL 41-80distribute G-REX 100M 2 2000 mL between flasks >80 Freeze fragments inCS10 after 15 minute pre- incubation

-   -   5. Advanced Preparation: Day 11 (Prepared up to 24 hours in        advance)        -   5.1. Prepared 6 L of CM2 with GlutaMax. Used reference            laboratory procedures for “Preparation of media for PreREP            and REP” for CM2 instructions”. Warmed at 37° C. 1 hour            prior to use.        -   5.2. Thawed IL-2 aliquots: Removed IL-2 aliquots from            freezer and placed at 4° C.    -   6. Harvest TIL (Day 11)        -   6.1. Carefully removed G-REX-100M flasks from incubator and            placed in BSC2. Were careful to not disturb the cells on the            bottom of the flask.        -   6.2. Using GatherRex or peristaltic pump aspirated ˜900 mL            of cell culture supernatant from flask(s).        -   6.3. Resuspended TIL by gently swirling flask. Observed that            all cells have been liberated from the membrane.        -   6.4. Using peristaltic pump or GatherRex transferred the            residual cell suspension to an appropriately sized blood            transfer pack (300-1000 mL). Was careful to not allow the            fragments to be transferred to the blood transfer pack.        -   6.5. Spiked the transfer pack with a 4″ plasma transfer set            (ensure clamp is closed).        -   6.6. Massaged the pack to ensure the cell suspension was            well mixed and using a 3 mL syringe, removed 1 mL TIL            suspension for cell counts. Clamped the tubing and recapped            female luer connector with a new sterile luer cap.        -   6.7. Placed the transfer pack into a plastic zip top bag and            replaced into the incubator until ready to use.    -   7. Media preparation        -   7.1. Allowed media to warm at 37° C. for >1 hr.        -   7.2. Added 3 mL of 6×10⁶IU/mL stock rhIL-2 to 6 L CM2 to            reach a final concentration of 3,000 IU/mL rhIL-2. Label as            “complete CM2”.        -   7.3. Sterile welded a 4″ plasma transfer set with female            luer to a 1 L Transfer pack.        -   7.4. Transferred 500 mL complete CM2 to a 1 L transfer pack.            Detached fluid transfer set or syringe and attached a            sterile luer plug to the female luer port.        -   7.5. Spiked the pack with a sample site coupler.        -   7.6. Using a 1.0 mL syringe with needle drew up 150 μL of 1            mg/mL anti-CD3 (clone OKT3) and transferred to 500 mL            “complete CM2” through sample site coupler. Drew back on the            syringe to ensure all reagent was flushed from the line.            Stored at 37° C. until use.    -   8. Flask preparation        -   8.1. Transferred 4.5 L “complete CM2” to a G-REX-500M flask            using the graduations on the flask for reference.        -   8.2. Placed flask into 37° C. incubator until ready.    -   9. Thaw irradiated feeders        -   9.1. Utilized 5.0×10⁹ allogenic irradiated feeders from two            or more donors for use.        -   9.2. Removed feeders from LN2 freezer and placed in a            biohazard transport bag.        -   9.3. With feeder bags in the biohazard transport bag, thawed            feeders in 37° C. incubator or bead bath. Kept bags static            and submerged. Removed feeders from bath when almost            completely thawed but still cold.        -   9.4. Sprayed or wiped feeder bags with 70% EtOH and place in            BSC2. Added each feeder bag directly to the open G-Rex 500M            to assure sufficient number of irradiated cells (5×10⁹            cells, +/−20%).        -   9.5. Closed both clamps on a fenwal Y type connector with            male luer lock.        -   9.6. Spiked each feeder bag with a leg of the Y connector.        -   9.7. Removed 1 L transfer pack with 500 mL “complete            CM2”+OKT3 and transferred to BSC.        -   9.8. Aseptically attached a 60 mL syringe to a 3 way            stopcock, and aseptically attached the transfer pack to the            male end of the stopcock.        -   9.9. Aseptically attached the Y connector to the 3 way            stopcock.        -   9.10. Drew the entire contents of the feeder bags into the            syringe, recorded the volume, and dispensed 5.0×10⁹            allogenic irradiated feeders into the transfer pack.        -   9.11. Clamped and detached transfer pack from apparatus, and            plug female luer lock with a new sterile luer plug.        -   9.12. Using a needle and 3 mL syringe pulled 1 mL for cell            counts from the sample site coupler.        -   9.13. When +/−10% of the target cell number (5.0×10⁹) was            reached with >70% viability, proceeded.        -   9.14. When less than 90% of the target cell number (5.0×10⁹)            was reached with >70% viability thawed another bag and            repeated 7.9.4-7.9.12. When greater than 110% of the target            cell number was achieved, calculated the proper volume            required for desired cell dose and proceeded.    -   10. Co-culture TIL and feeders in G-REX 500M flask        -   10.1. Removed the G-REX 500M flask containing prepared media            from the incubator and placed in the BSC2.        -   10.2. Attached feeder transfer pack to G-REX-500M and            allowed contents of the bag to drain into the 500M.        -   10.3. Removed TIL suspension from the incubator and placed            in the BSC.        -   10.4. Calculated volume of TIL suspension to add to achieve            200×10⁶ total viable cells.            (TVC/mL)/200×10⁶=mL        -   10.5. When TIL were between 5-200×10⁶ total viable cells,            added all TIL (total volume) to the G-REX-500M. When TIL            count was greater than 200×10⁶ total viable cells, added            calculated volume necessary for 200×10⁶ TIL to be            distributed to an individual G-REX-500M. Remaining TIL were            spun down and frozen in at least two cryovials at up to            10⁸/mL in CS10, labeled with TIL identification and date            frozen.        -   10.6. Placed the G-REX-500M in a 37° C., 5% CO₂ incubator            for 5 days.    -   11. Advanced preparation: Day 16-18        -   11.1. Warmed 1 10 L bag of AIM V for cultures initiated with            less than 50×10⁶ TIL warmed 2 for those initiated with            greater than 50×10⁶ TIL at 37° C. at least 1 hr or until            ready to use.    -   12. Perform TIL cell count: Day 16-18        -   12.1. Removed G-REX-500M flask from incubator and placed in            BSC2. Were careful not to disturb the cell culture on the            bottom of the flask.        -   12.2. Aseptically removed 4 L of cell culture media from the            G-REX-500M flask and placed into a sterile container.        -   12.3. Swirled the G-REX-500M until all TIL had been            resuspended from the membrane.        -   12.4. Using GatherRex or peristaltic pump transferred cell            suspension to a 2 L transfer pack. Retained the 500M flask            for later use. Sealed the port with the sample site coupler            to avoid loss of TILs.        -   12.5. Spiked the transfer pack with a sample site coupler            and using a 3 mL syringe and needle removed 2×1 mL            independent samples for a cell count.        -   12.6. Calculated the total number of flasks required for            subculture according to the following formula. Rounded            fractions up.            Total viable cells/1.0×10⁹=flask #    -   13. Prepare CM4        -   13.1. Prepared a 10 L bag of AIM-V for every two 500M flasks            needed. Warmed additional media as necessary.        -   13.2. For every 10 L of AIM-V needed, added 100 mL of            GlutaMAX to make CM4.        -   13.3. Supplemented CM4 media with rhIL-2 for a final            concentration of 3,000 IU/mL rhIL-2.    -   14. Split the cell culture        -   14.1. Using the graduations on the flask, gravity filled            each G-REX-500M to 5 L.        -   14.2. Evenly distributed the TIL volume amongst the            calculated number of G-REX-500 Ms.        -   14.3. Placed flasks in a 37° C., 5% CO₂ incubator until            harvest on Day 22 of REP.    -   15. Advanced Preparation: Day 22-24        -   15.1. Prepared 2 L of 1% HSA wash buffer by adding 40 mL of            25% HSA to each of two 1 L bags of PlasmaLyte A 7.4. Pool            into a LOVO ancillary bag.        -   15.2. Supplemented 200 mL CS10 with IL-2 @ 600 IU/mL.        -   15.3. Pre-cooled four 750 mL aluminum freezer canisters at            4° C.    -   16. Harvest TIL: Day 22-24        -   16.1. Removed the G-REX-500M flasks from the 37° C.            incubator and placed in the BSC2. Were careful to not            disturb the cell culture on the bottom of the flask.        -   16.2. Aspirated and discarded 4.5 L of cell culture            supernatant from each flask.        -   16.3. Swirled the G-REX-500M flask to completely resuspend            the TIL.        -   16.4. Weighed the 3-5 L bioprocess bag prior to use.        -   16.5. Using GatherRex or peristaltic pump, harvested TIL            into the bioprocess bag.        -   16.6. Mixed bag well and using a 3 mL syringe take 2×2 mL            samples from the syringe sample port for cell counting.        -   16.7. Weighed the bag and found the difference between the            initial and final weight. Used the following calculation to            determine the volume of cell suspension.            Net weight of cell suspension (mL)/1.03=volume (mL)    -   17. Filter TIL and prepare LOVO Source bag        -   17.1. Placed the bag containing cell culture into the BSC2.        -   17.2. Placed a 170 μm blood filter into the BSC2 and closed            all clamps.        -   17.3. Sterile welded a source leg of the filter to the cell            suspension.        -   17.4. Weighed a new appropriately sized bioprocess bag (this            was referred to as the LOVO source bag).        -   17.5. Sterile welded the terminal end of the filter to the            LOVO source bag.        -   17.6. Elevated the cell suspension by hanging cells on an IV            pole to set up a gravity-flow transfer of cells.        -   Note: (Did not allow the source bag to hang from the            filtration apparatus.)        -   17.7. Opened all necessary clamps and allowed TIL to drain            from the cell suspension bag through the filter and into the            LOVO source bag.        -   17.8. Once all cells were transferred to the LOVO source            bag, closed all clamps and sealed the LOVO source bag tubing            to remove filter.        -   17.9. Weighed the LOVO source bag and calculate volume.        -   17.10. The LOVO source bag was ready for the LOVO.        -   17.11. Removed the LOVO final product bag from the            disposable kit by sealing the tubing near the bag.    -   18. Formulate TIL 1:1 in cold CS10 supplemented with 600 IU/mL        rhIL-2        -   18.1. Calculated required number of cryobags needed.            (volume of cell product×2)/100=number of required bags            (round down)        -   18.2. Calculated the volume to dispense into each bag.            (volume of cell product×2)/number of required bags=volume to            add to each bag        -   18.3. Aseptically transferred the following materials in            Table 6 to the BSC.

TABLE 6 Materials for TIL cryopreservation. Minimum Item QuantityIn-Process Label Cell product 1 Lot# Aluminum freezer cassette (750 ml)1 n/a Cold CS10 + IL-2 @600 IU/mL As Needed Lot# Cell Connect CC1 device1 n/a 750 mL cryobags calculated Label aliquots 1 - largest# 100 mLsyringe #cryobags + 1 n/a 3 way stopcock 1 n/a Cryovials 5 TILCryo-product satellite vials

-   -   19. TIL formulation        -   19.1. Closed all clamps on Cell Connect CC1.        -   19.2. To the cell connect device aseptically attached the            LOVO final product, CS10 bag luer lock and the appropriate            number of cryobags. Replaced the 60 mL syringe with a 100 mL            syringe.        -   19.3. The amount of CS10 volume needed was equivalent to the            volume of the LOVO final product bag.        -   19.4. Opened the stopcock pathway and unclamp the line            between the LOVO final product bag and syringe to pull CS10            into the syringe, reclamp CS10 path. Unclamped pathway to            the cell bag to push CS10 into the LOVO final product bag.            Used the syringe to measure the volume added to the LOVO            final product bag. Repeated as necessary using a new syringe            until desired amount of CS10 is transferred.        -   19.5. Mixed LOVO final product bag by inversion.        -   19.6. Replaced 100 mL syringe        -   19.7. Opened clamps on 750 mL cryobags one at a time        -   19.8. Only opened clamps that are directly associated with            the formulated product and the cryobag in use.        -   19.9. Used the 100 mL syringe to measure the volume of            formulated product leading to the cryobag.        -   19.10. Transferred 100 mL of formulated product into each            cryobag.        -   19.11. After addition to each bag pulled back on the syringe            to remove all air bubbles from cryobags and reclamped the            associated line.        -   19.12. On the final bag pull back a 10 mL retain for QC            testing.        -   19.13. Sealed each cryobag, leaving as little tubing as            possible.        -   19.14. Removed the syringe containing the retained sample            and transferred to a 50 mL conical tube; transferred 1.5 ml            into individual cryovials and froze into a controlled rate            freezer.        -   19.15. Transferred sealed bags to 4° C. while labels were            prepared.        -   19.16. Labeled each cryobag with product description, name            and date, volume, cell count, and viability.        -   19.17. Placed each cryobag into pre-cooled aluminum freezer            canisters.    -   20. Cryopreservation of TIL using Control Rate Freezer (CRF)        -   20.1. Followed standard procedure for the controlled rate            freezer.        -   20.2. After using the CRF, stored cryobags in liquid            nitrogen (LN2).    -   21. Determined expected results and measure acceptance criteria.

Example 2: Process Run on 8 Patient Tumors

The process of Example 1 was run using 8 patient tumors to produce 8batches of TILs. Good recovery from culture, viability, cell counts,CD3⁺ (indicating the % T cell content) and IFN-gamma (IFN-g or IFN-γ)release were obtained, as shown in Table 7 below and in FIG. 7 throughFIG. 10.

TABLE 7 Results of Testing of Identity, Potency, and Viability/Recoveryof the Process of Example 1. IFNg (pg/1e6 Cells/mL cells/ (Viable + %Recovery % Via- 24 hr) CD3 (%) Nonviable) Fresh/Lovo bility M1061T 457095.3 1.27E+08 103 88.1 M1062T 3921 99.7 1.65E+08 89 84.5 M1063T 558798.7 1.51E+08 112 82.1 M1064T 619 84.5 1.75E+08 83 86.8 M1065T 1363 96.83.42E+07 128 76.4 EP11001T 4263 90.4 1.82E+08 92 77.9 M1056T 6065 94.22.11E+08 85 84.8 M1058T 1007 99 2.72E+08 89 87.5

Example 3: Scalability of Modified Til Process

The studies presented here were performed in a process development (PD)lab, and subsequently, a process qualification (PQ) study utilizingengineering runs was performed in the GMP clean room suite at amanufacturing facility. Three PQ/engineering runs were completed in theGMP facility clean room according to a qualification protocol, and abatch record based on the PD studies presented here. Acceptance criteriafor the engineering runs were set prospectively. The PQ study is furthersummarized below, and test results obtained for the engineering batchesare provided in the following sections.

The number of cells generated from pre-rapid expansion protocol(pre-REP) cultures often exceeded 100×10⁶ viable cells. In addition,including a freeze-thaw cycle between the Pre-REP and REP culture stepsreduced the viable cell yield. By eliminating the in-processcryopreservation step, the REP could be reliably and regularly initiatedwith an increased number of TIL. This change allowed the duration of theREP to be decreased by a proportional amount of cell doubling times toroughly 11 days without impacting cell dose. In addition, the reducedculture time from activation to harvest results in a product that isless differentiated and potentially better able to persist in-vivo (Tran2008).

The PD study validated the initiation of the REP culture with up to200×10⁶ cells with a fixed number of feeder cells. The optimal time toharvest the REP culture was then evaluated over 9 to 14 days. Cultureswere seeded at feeder to TIL ratios ranging from 100:1 to 25:1.Optimization of harvest time was determined by measuring total cellcount, viability, immunophenotype, media consumption, metaboliteanalysis, interleukin-2 (IL-2) analysis, and the functional analysesdescribed below.

Immunophenotyping of cells at the end of the REP culture was evaluatedon the basis of the markers listed in Table 8 below. The phenotypicactivation and differentiation state of the cells was evaluated.Statistical differences in phenotype were not observed among any of theexperimental conditions.

TABLE 8 Markers of Activation and Differentiation Assayed on ProcessOptimization Cultures. Target Label for Detection Clone Panel 1 TCRab(i.e., TCRα/β) PE/Cy7 IP26 CD57 PerCP-Cy5.5 HNK-1 CD28 PE CD28.2 CD4FITC OKT4 CD27 APC-H7 M-T271 CD56 APC N901 CD8a PB RPA-T8 Panel 2 CD45RAPE/Cy7 HI100 CD3 PerCP/Cy5.5 SP34-2 CCR7 PE 150503 CD8 FITC HIT8 CD4APC/Cy7 OKT4 CD38 APC HB-7 HLA-DR PB L243 Panel 3 CD137 PE/Cy7 4B4-1 CD3PerCP/Cy5.5 SP34-2 Lag3 PE 3DS223H CD8 FITC HIT8 CD4 APCCy7 OKT4 PD1 APCEH12.2H7 Tim-3 BV421 F38-2E2

Abbreviations: PE/Cy7=Phycoerythrin: Cy-7 Tandem Conjugate;PerCP-Cy5.5=Peridinin-chlorophyll-protein Complex:CY5.5 Conjugate;PE=Phycoerythrin; FITC=Fluorescein Isothiocyanate Conjugate;APC-H7=Allophycocyanin:H7 Tandem Conjugate; APC=Allophycocyanin;PB=Pacific Blue™

Media consumption and metabolite production remained within tolerablelimits for all conditions tested; and IL-2 levels remained greater than150 IU/mL of culture supernatant (data not shown).

Tumor cell killing by T-cells is understood to be mediated by activationof the T-cell receptor on the effector T-cell in response to peptidespresented on the surface of tumor cells. Ex vivo expanded T-cells mustretain the ability to be activated and proliferate in response to TCRactivation if they are to persist in vivo upon infusion and mediatetumor regression.

To assess the activation potential of the cultured cells, TIL harvestedat different time points were reactivated with irradiated allogeneicPBMC loaded with OKT3. TIL cultures were harvested after 7 days andassayed for fold-expansion. The results of this study are summarized inTABLE 9.

TABLE 9 Summary of Results: Proliferation of Post-REP TIL UponRe-culture with OKT3 Loaded Allogeneic PBMC. P-value Harvest DayFold-Expansion SD (Student ‘t’test) Experiment 1 Day 9 43 6.088 NA Day10 48 3.105 NA Day 11 71 11.137 0.135 Day 14 60 6.995 Experiment 2 Day 944 6.276 NA Day 10 27 4.762 NA Day 11 72 18.795 0.045 Day 14 41 7.050Experiment 3 Day 9 54 5.810 NA Day 10 54 9.468 NA Day 11 65 1.674 0.071Day 14 50 8.541

This study demonstrated that the potential for TIL to be activated inthis assay increased with each day of culture through Day 11 (harvestDays 9-11). Cells harvested on Day 11 of the modified process performedsimilarly to control TIL maintained in culture for 14 days similar tothe current process.

These studies demonstrated the scalability of the modified TIL processand established an acceptable range of seeding ratios of TIL to feedercells. In addition, the growth characteristics were found to persistthrough Day 14 of culture, while culture conditions remained optimalthrough Day 11. The conditions tested showed no measurable effect on TILphenotype. Cells harvested on REP culture Day 11 demonstrated the bestability to respond to reactivation while the cell culture conditionsremained within tolerances. These changes were adopted and validated atfull scale with the culture split occurring on Day 5 and harvest on Day11.

Engineering runs were implemented at the process development facility inorder to gain experience in manufacturing and testing the TIL productprior to the GMP manufacturing of autologous TIL product foradministration to patients. The manufacturing procedure used for theengineering runs was at the same scale as that to be used in themanufacturing of GMP TIL product. Experience in growing TIL from varioustypes of tumors including metastases of melanoma, breast, head and necksquamous cell carcinoma (HNSCC), cervical carcinoma, and lung cancer hasdetermined that the dissection and outgrowth of TIL from metastatictumor samples is similar for these cancers (Sethuraman 2016, JITC P42).Because the initial isolation of tumor fragments and outgrowth oflymphocytes appears to be similar between tumor histologies, theseengineering runs are sufficient to qualify the process for theproduction of TIL from HNSCC, cervical and melanoma tumors.

Table 10 shows the source and characteristics of tumor samples used forthe engineering runs.

TABLE 10 Tumor Samples Tested for Engineering Runs. Tumor EngineeringSample Engineering Run 1 Engineering Run 2 Run 3 Patient ID 1001185600-D455 40231 Source Biotheme Research BioOptions Moffitt Tissue Lung,Left Breast, ERPR+Her2− Melanoma Date Jan. 5, 2017 Jan. 12, 2017 Jan.26, 2017 Processed

Release testing of the three engineering runs of TIL at the processdevelopment facility was completed (Table 11) as described below.Product was tested on Day 16 and Day 22. IFN-γ secretion was alsodetermined for the three engineering runs (Table 12) as detailedelsewhere.

TABLE 11 Product Release Test Results for Engineering Runs at ProcessDevelopment Facility. Test Acceptance Engineering Runs Parameter MethodCriteria Run 1 Run 2 Run 3 Day 16 Sterility* BacTAlert No Grwoth Nogrowth No growth No growth Mycoplasma PCR Negative from NegativeNegative Negative Day 7 split Day 22 Viability (%) AOPI ≥70% 82.3%85.13%  84.6% Total Viable AOPI Report results 2.6 × 10¹⁰ 1 × 10¹⁰ 1.4 ×10¹¹ Cells Sterility Gram Stain Negative Negative Negative NegativeSterility Final BacT/Alert No Growth Negative Negative Pending Product*% CD45⁺CD3⁺ Flow ≥90% 99.3% 96.3% 99.8% cytometey Endotoxin EndoSafe≤0.7 EU/mL <0.5 EU/mL <0.5 EU/mL <0.5 EU/mL Mycoplasma PCR NegativeNegative Negative Negative Final Product Appearance Visual Intact bagwith no Intact bag, no Intact bag, no Intact bag, no Inspection visibleclumps visible clumps visible clumps visible clumps *Final sterilityresults for Day 16 and Day 22 are not available until after finalproduct release for shipment. The gram stain results from Day 22 areused for sterility shipment release.

TABLE 12 Additional Functional Characterization: Measurement of IFN-γSecretion. Functional Expected Engineering Run Characterization MethodResults Run 1 Run 2 Run 3 IFN-γ Stimulation with ELISA >2 standard 3085+/− 182 2363 +/− 437 pending anti-CD3, CD28, CD137 deviations over(pg/million cells) non-stimulated IFN-γ Non-stimulated ELISA Notapplicable 34 +/− 5  27 +/− 10 pending (pg/million cells)

In conclusion, the data from the engineering runs demonstrate that TILdrug product can be manufactured for the purpose of autologousadministration to patients.

Example 4: Lymphodepletion

Cell counts can be taken at day 7 and prior to lymphodepletion. Thefinal cell product included up to approximately 150×10⁹ viable cellsformulated in a minimum of 50% HypoThermosol™ in Plasma-Lyte A™(volume/volume) and up to 0.5% HSA (compatible for human infusion)containing 300 IU/mL IL2. The final product was available foradministration in one of two volumes for infusion:

-   -   1) 250 mL (in a 300-mL capacity infusion bag) when the total TIL        harvested are ≤75×10⁹    -   OR    -   2) 500 mL (in a 600-mL capacity infusion bag) when the total TIL        harvested are <150×10⁹

The total number of cells that could be generated for the final TILinfusion product for each patient due to patient-to-patient variation inT-cell expansion rates during the REP step cannot be predicted. A lowerlimit of cells on day 3, 4, 5, 6, 7 of the 3 to 14-day REP is set basedon the minimum number of cells needed in order to make a decision tolymphodeplete the patient using the cyclophosphamide plus fludarabinechemotherapy regimen. Once we have begun lymphodepletion based on thisminimal attained cell number, we are committed to treating the patientwith the available number of TIL we generate in the REP by any of days 3to 14, and in many cases day 7. The upper limit of the range forinfusion (150×10⁹ viable cells) is based on the known published upperlimit safely infused where a clinical response has been attained.Radvanyi, et al., Clin Cancer Res 2012, 18, 6758-6770.

Example 5: Process 2A—Day 0

This example describes the detailed day 0 protocol for the 2A processdescribed in Examples 1 to 4.

Preparation.

-   -   1. Confirmed Tumor Wash Medium, CM1, and IL-2 are within        expiration date.    -   2. Placed CM1 (cell media 1) in incubator.

Method.

-   -   1. Cleaned the biological safety cabinet (BSC).    -   2. Set up in-process surveillance plates and left in biosafety        cabinet for 1-2 hours during procedure.    -   3. Placed the TIL media CM1 in the biological safety cabinet.    -   4. Prepared TIL media CM1 containing 6000 IU/mL IL-2:        -   4.1. 1 L CM1        -   4.2. 1 ml IL-2 (6,000,000 IU/mL)        -   4.3. Placed 25 ml of CM1+IL2 into 50 ml conical to be used            for fragments when adding to G-REX.        -   4.4. Placed in 37° C. incubator to pre-warm    -   5. Wiped G-REX 100MCS package with 70% alcohol and place in        biosafety cabinet. Closed all clamps except filter line.    -   6. Performed Acacia pump calibration.    -   7. Attached the red line of G-REX 100MCS flask to the outlet        line of the acacia pump boot.    -   8. Attached pumpmatic to inlet line of pump boot and placed in        bottle with media. Released clamps to pump boot.    -   9. Pumped remaining 975 ml of pre-warmed CM1 containing 6,000        IU/ml of IL-2 in each G-REX 100MCS bioreactor.    -   10. Heated seal red line, disconnect from pump boot.    -   11. Placed label on G-REX.    -   12. Placed G-REX 100MCS in incubator until needed.

Tissue Dissection

-   -   1. Recorded the start time of tumor processing.    -   2. Transferred Tumor Wash Medium to BSC.    -   3. Placed 5 100 mm petri dishes in biosafety cabinet, 3 for        washes, 1 for holding and 1 for unfavorable tissue. Labeled        dishes accordingly. Unfavorable tissue was indicated by yellow        adipose tissue or necrotic tissue.    -   4. Placed three 6 well plates into biosafety cabinet.    -   5. Pipetted 3-5 mL of Tumor Wash Medium into each well of one        six well plates for excess tumor pieces.    -   6. Pipetted 50 mL of Tumor Wash Medium to wash dishes 1-3 and        holding dish.    -   7. Placed two 150 mm dissection dishes into biosafety cabinet.    -   8. Placed 3 sterile 50 mL conical tubes into the BSC.    -   9. Labeled one as forceps tumor wash medium, the second as        scalpel tumor wash medium, and third for Tumor wash medium used        in for lid drops.    -   10. Added 5-20 mL of tumor wash medium to each conical. The        forceps and scalpels were dipped into the tumor wash media as        needed during the tumor washing and dissection process.    -   11. Placed scalpel and forceps in appropriate tubes.    -   12. Using long forceps removed the tumor(s) from the Specimen        bottle and transferred to the Wash 1 dish.    -   13. Incubated the tumor at ambient in the Wash 1 dish for ≥3        minutes.    -   14. During the incubation, re-labeled the Specimen bottle        “Bioburden” and stored at 2-8° C. until the final harvest or        further sterility testing is required.    -   15. Using forceps transferred the tumor to the Wash 2 dish.    -   16. Incubated the tumor at ambient in the Wash 2 dish for ≥3        minutes.    -   17. During the incubation, using a transfer pipette, added        approximately 4 evenly-spaced, individual drops of Tumor Wash        Medium to each circle of the 6 well plate lids designated as        Tumor Fragments dishes.    -   18. Using forceps transferred the tumor to the Wash 3 dish.    -   19. Incubated the tumor at ambient in the Wash 3 dish for ≥3        minutes.    -   20. The 150 mm dish lid was used for dissection. Placed a ruler        underneath.    -   21. Using forceps transferred the tumor to the Dissection dish,        measured and recorded the length of the tumor.    -   22. Took photograph of tumor.    -   23. Performed an initial dissection of the tumor pieces in the        Dissection dish into intermediate pieces taking care to conserve        the tumor structure of each intermediate piece.    -   24. Transferred any intermediate tumor pieces not being actively        dissected into fragments to the tissue holding dish to ensure        the tissue remained hydrated during the entire dissection        procedure.    -   25. Worked with one intermediate tumor piece at a time,        carefully sliced the tumor into approximately 3×3×3 mm fragments        in the Dissection Dish, using the rule underneath the dish for        reference    -   26. Continued dissecting fragments from the intermediate tumor        piece until all tissue in the intermediate piece had been        evaluated.    -   27. Selected favorable fragments and using a transfer pipette        transferred up to 4 favorable fragments into the wash medium        drops in one circle in the Tumor Fragments dish. Using a        transfer pipette scalpel or forceps, transferred, as much as        possible of the unfavorable tissue and waste product to the        Unfavorable Tissue dish to clear the dissection dish. All        remaining tissue was place into one of the wells of the six-well        plate. (Unfavorable tissue was indicated by yellow adipose        tissue or necrotic tissue.)    -   28. Continued processing by repeating step 23-26 for the        remaining intermediate tumor pieces, working one intermediate        piece at a time until the entire tumor had been processed.        (Obtained a fresh scalpel or forceps as needed, to be decided by        processing technician.)    -   29. Moved fragment plates toward rear of hood.    -   30. Using transfer pipette, the scalpel, or the forceps,        transferred up to 50 of the best tumor fragments to the 50 mL        conical tube labeled tumor fragments containing the CM1.    -   31. Removed floaters from 50 mL conical with transfer pipet.        Recorded number of fragments and floaters.    -   32. Removed all unnecessary items from hood, retaining the        favorable tissue plates if they contain extra fragments. Wiped        hood with alcohol wipe.    -   33. Removed G-REX 100MCS from incubator, wipe with 70% alcohol        and place in biosafety cabinet.    -   34. Swirled conical with tumor fragments and poured the contents        on the 50 ml conical into the G-Rex 100MCS flask    -   35. If one or more tumor fragments transferred to the G-Rex 100M        flask float, obtained one additional tumor fragment when        available from the Favorable Tissue Dish and transfer it to the        G-Rex 100M flask.    -   36. Recorded incubator # (s) and total number of fragments added        to each flask.    -   37. Placed the G-REX 100M bioreactor in 37° C., 5% CO₂ incubator    -   38. Any unused tumor were placed in 100 mL of HypoThermosol and        delivered to the laboratory.    -   39. Recorded the stop time of tumor processing.    -   40. Discarded any un-used TIL complete media containing IL-2 and        any un-used aliquots of IL-2.    -   41. Cleaned biological safety cabinet.    -   42. Placed the Bioburden sample in the proper storage        conditions.    -   43. Recorded data.    -   44. Saved the picture as file specimen ID # Tumor process Date        to the prepared patient's file.    -   45. Ordered and ensured delivery of settle plates to the        microbiology lab.

Example 6: Process 2A—Day 11

This example describes the detailed day 11 protocol for the 2A processdescribed in Examples 1 to 4.

Prior Preparation.

-   -   1. Day before processing:        -   1.1. CM2 could be prepared the day before processing            occurred. Place at 4° C.    -   2. Day of processing.        -   2.1. Prepared the feeder cell harness.            -   2.1.1. Closed all clamps on a CC2 and 4S-4M60 connector                sets.            -   2.1.2. Sterile welded 4 spikes of 4S-4M60 harness to the                spike line on the CC2 removing the spike.            -   2.1.3. Set aside for feeder cell pooling.        -   2.2. Prepared 5 mL of cryopreservation media per            CTF-FORM-318 and place at 4° C. until needed.

Clean Room Environmental Monitoring—Pre-Processing

-   -   1. Recorded clean room information.    -   2. Biosafety Cabinets (BSC) were cleaned with large saturated        alcohol wipes or alcohol spray.    -   3. Verified Particle Counts for 10 minutes before beginning        processing.    -   4. Set up in-process surveillance plates and left in biosafety        cabinet for 1-2 hours during procedure.

Prepare G-Rex 500MCS Flask:

-   -   1. Using 10 mL syringe aseptically transferred 0.5 mL of IL-2        (stock is 6×10⁶ IU/mL) for each liter of CM2 (cell media 2) into        the bioprocess bag through an unused sterile female luer        connector.    -   2. Used excess air in the syringe to clear the line, drew up        some media from the bag and expel back into back. This ensured        all the IL-2 has been mixed with the media. Mixed well.    -   3. Opened exterior packaging and place G-Rex 500MCS in the BSC.        Closed all clamps on the device except large filter line.    -   4. Sterile welded the red harvest line from the G-Rex 500MCS to        the pump tubing outlet line.    -   5. Connected bioprocess bag female luer to male luer of the Pump        boot.    -   6. Hung the bioprocess bag on the IV pole, opened the clamps and        pump 4.5 Liters of the CM2 media into the G-Rex 500MCS. Cleared        the line, clamp, and heat seal.    -   7. Retained the line from pump to media. It was used when        preparing feeder cells.    -   8. Placed G-Rex 500MCS in the incubator.

Prepare Irradiated Feeder Cells

-   -   1. Sealed and removed spike(s) from IL TP. Clamped both lines.    -   2. Recorded the dry weight of a 1 L transfer pack (TP).    -   3. Sterile welded the 1 L transfer pack to the acacia pump boot        ˜12″ from bag.    -   4. The other end of the pump tubing was still connected to the        10 L labtainer.    -   5. Pumped 500 mL CM2 by weight into the TP.    -   6. Closed clamp and sealed close to weld joint.    -   7. Placed in incubator.    -   8. Verified and Logged out feeder cell bags.    -   9. Recorded feeder lot used.    -   10. Wiped bags with alcohol.    -   11. Placed in zip lock bags.    -   12. Thawed feeder cells in the 37° C. (+/−1° C.) water bath.        Recorded temperature of water bath.    -   13. Removed and dried with gauze.    -   14. Passed feeder cells through pass thru into Prep Room.    -   15. Transferred to BSC in Clean Room.    -   16. Using the previously prepared feeder harness, welded the 1 L        TP with media to one of the unused lines on the sample port side        of the 3 way stopcock as close as possible to the seal junction        loosing as little tubing as possible.    -   17. Put feeder harness into BSC.    -   18. Spiked each of the 3 feeder bags with the spike from the        feeder harness into the single port of the feeder bag.    -   19. Rotated the stopcock valve so the 1 L TP is in the “OFF”        position.    -   20. Working with one bag at a time, opened the clamps on the        line to the feeder bag, expel air in syringe and draw the        contents of the feeder bag into the syringe. Expelled air from        syringe helped in recovering cells. Closed clamp to feeder bag.    -   21. Recorded the volume recovered of thawed feeder cells in each        bag.    -   22. Rotated the stopcock valve so that the feeder bag is in the        “OFF” position    -   23. Opened the clamp on the TP and dispense the contents of the        syringe into the TP.    -   24. Ensured the line has been cleared and re-clamp the TP. You        may have had to draw some air into syringe from TP for use in        clearing the line.    -   25. Mixed the cells well.    -   26. Closed clamp to feeder bag.    -   27. Rotated stopcock so syringe port is in the “OFF” position.        Disconnected the 60 mL syringe from the stopcock.    -   28. Replaced with new syringe for each feeder bag.    -   29. Left syringe on after final bag.    -   30. Mixed final feeder formulation well.    -   31. Rotated stopcock so feeder cell suspension is in the “OFF”        position.    -   32. Mixed cells cell and using a 5 mL syringe and needless port,        rinsed port with some cell solution to ensure accurate sampling        and remove 1 ml of cells, placed into tube labeled for counting.    -   33. Repeated with second syringe. These two independent samples        each had a single cell count performed.    -   34. Turned stopcock so feeder suspension is in the “OPEN”        position and using the 60 ml syringe attached to harness        expelled air into the TP to clear the line.    -   35. Removed syringe and covered luer port with a new sterile        cap.    -   36. Heated seal the TP close to weld joint, removed the harness.    -   37. Recorded mass of transfer pack with cell suspension and        calculated the volume of cell suspension.    -   38. Placed in incubator.    -   39. Performed a single cell counts on the feeder cell sample and        record data and attach counting raw data to batch record.    -   40. Documented the Cellometer counting program.    -   41. Verified the correct dilution was entered into the        Cellometer.    -   42. Calculated the total viable cell density in the feeder        transfer pack.    -   43. If cell count was <5×10⁹, thawed more cells, count, and        added to feeder cells.    -   44. Re-weighed feeder bag and calculated volume.    -   45. Calculated volume of cells to remove.

Addition of Feeder to G-REX

-   -   1. Sterile welded a 4″ transfer set to feeder TP.    -   2. In the BSC attached an appropriately sized syringe to the        female luer welded to the feeder transfer pack.    -   3. Mixed cells well and removed the volume calculated in step 40        or 41 to achieve 5.0×10⁹ cells. Discarded unneeded cells.    -   4. Using a 1 mL syringe and 18 G needle draw up 0.150 mL of        OKT3, removed needle and transferred to the feeder TP through        the female luer.    -   5. Rinsed tubing and syringe with feeder cell and mixed bag        well. Cleared the line with air from syringe.    -   6. Removed the G-Rex 500MCS from the incubator, wiped with        alcohol wipes and placed beside the SCD.    -   7. Sterile welded the feeder bag to the red line on the G-Rex        500MCS. Unclamped the line and allowed the feeder cells to flow        into the flask by gravity.    -   8. Ensured the line has been completely cleared then heat sealed        the line close to the original weld and removed the feeder bag.    -   9. Returned the G-Rex 500MCS to the incubator and recorded time.

Prepare TIL: Record Time Initiation of TIL Harvest

-   -   1. Carefully removed G-Rex 100MCS from incubator and closed all        clamps except large filter line.    -   2. Welded a 1 L transfer pack to the redline on the G-REX        100MCS.    -   3. Closed clamp on a 300 ml TP. Heat seal ˜12 inches from the        bag removing the spike. Recorded dry weight/mass.    -   4. Sterile welded the 300 mL transfer pack to the cell        collection line on the 100MCS close to the heat seal. Clamped        the line.    -   5. Released all clamps leading to the 1 L TP.    -   6. Using the GatheRex transferred ˜900 mL of the culture        supernatant to the 1 L transfer pack. Gatherex stopped when air        entered the line. Clamped the line and heat seal.    -   7. Swirled the flask until all the cells had been detached from        the membrane. Checked the membrane to make sure all cells are        detached.    -   8. Tilted flask away from collection tubing and allowed tumor        fragments to settle along edge.    -   9. Slowly tipped flask toward collection tubing so fragments        remain on opposite side of flask.    -   10. Using the GatheRex transferred the residual cell suspension        into the 300 mL transferred pack avoiding tumor fragments.    -   11. Rechecked that all cells had been removed from the membrane.    -   12. If necessary, back washed by releasing clamps on GatheRex        and allowed some media to flow into the G-Rex 100MCS flask by        gravity.    -   13. Vigorously tapped flask to release cells and pumped into 300        ml TP.    -   14. After collection was complete, closed the red line and heat        seal.    -   15. Heated seal the collection line leaving roughly the same        length of tubing as when dry weight was recorded.    -   16. Recorded mass (including dry mass) of the 300 ml TP        containing the cell suspension and calculated the volume of cell        suspension.    -   17. In the BSC spike the 300 mL TP with a 4″ plasma transferred        set. Mixed cells well. Aseptically attached a 5 mL syringe draw        1 mL, placed in cryo vial. Repeated with second syringe. These        were used for cell counting, viability.    -   18. Re-clamped and replaced luer cap with new sterile luer cap.    -   19. Placed in incubator and recorded time place in incubator.    -   20. Performed a single cell count on each sample and recorded        data and attach counting raw data to batch record.    -   21. Documented the Cellometer counting program.    -   22. Verified the correct dilution was entered into the        Cellometer.    -   23. If necessary adjusted total viable TIL density to ≤2×10⁸        viable cells.    -   24. Calculated volume to remove or note adjustment not        necessary.    -   25. In the BSC aseptically attached an appropriately sized        syringe to the 300 mL TP.    -   26. If required, removed the calculated volume of cells        calculated in the “Calculate volume to remove” table.    -   27. Clamped and heat sealed the 300 ml TP.    -   28. Transferred excess cells to an appropriately sized conical        tube and placed in the incubator with cap loosened for later        cryopreservation.    -   29. Removed the G-Rex 500MCS from the incubator and place beside        the SCD.    -   30. Sterile welded the 300 ml TP to the inlet line of the Acacia        pump.    -   31. Sterile welded the red line of the G-Rex 500MCS to the        outlet line of the Acacia pump.    -   32. Pumped cells into flask.    -   33. Ensured the line has been completely cleared then heat        sealed the red line close to the original weld.    -   34. Checked that all clamps on the G-Rex 500MCS were closed        except the large filter line.    -   35. Returned the G-Rex 500MCS to the incubator and record the        time placed in the G-Rex incubator.    -   36. Ordered and ensured delivery of settle plates to the        microbiology lab.        Cryopreservation of Excess

Calculated amount of freezing media to add to cells:

TABLE 13 Target cell concentration was 1 × 10⁸/ml A. Total cells removed(from step 15) mL B. Target cell concentration 1 × 10⁸ cells/mL Volumeof freezing media to add (A/B) mL

-   -   37. Spun down TIL at 400×g for 5 min at 20° C. with full brake        and full acceleration.    -   38. Aseptically aspirated supernatant.    -   39. Gently tapped bottom of tube to resuspend cells in remaining        fluid.    -   40. While gently tapping the tube slowly added prepared freezing        media.    -   41. Aliquoted into appropriate size cryo tubes and record time        cells placed into −80° C.

Example 7: Process 2A—Day 16

This example describes the detailed day 16 protocol for the 2A processdescribed in Examples 1 to 4.

Clean Room Environmental Monitoring—Pre-Processing.

-   -   1. Biosafety Cabinets were cleaned with large saturated alcohol        wipes or alcohol spray.    -   2. Verified Particle Counts for 10 minutes before beginning        processing.    -   3. Set up in-process surveillance plates and left in biosafety        cabinet for 1-2 hour during procedure.

Harvest and Count TIL.

-   -   1. Warmed one 10 L bag of CM4 for cultures initiated with less        than 50×10⁶ TIL in a    -   37° C. incubator at least 30 minutes or until ready to use.    -   2. In the BSC aseptically attached a Baxter extension set to a        10 L Labtainer bag.    -   3. Removed the G-Rex 500MCS flask from the incubator and placed        on the benchtop adjacent the GatheRex. Checked all clamps were        closed except large filter line. Moved the clamp on the quick        connect line close to the “T” junction.    -   4. Sterile welded a 10 L Labtainer to the red harvest line on        the G-Rex 500MCS via the weldable tubing on the Baxter        extension.    -   5. Heat sealed a 2 L transfer pack 2″ below the “Y removing the        spike and recorded dry weight. Sterile welded the 2 L TP to the        clear collection line on the G-Rex 500MCS.    -   6. Set the G-Rex 500MCS on a level surface.    -   7. Unclamped all clamps leading to the 10 L Labtainer and using        the GatheRex transferred ˜4 L of culture supernatant to the 10 L        Labtainer.    -   8. Harvested according to appropriate GatheRex harvesting        instructions.    -   9. Clamped the red line and recorded time TIL harvest initiated.    -   10. GatheRex stopped when air entered the line. Clamped the red        line.    -   11. After removal of the supernatant, swirled the flask until        all the cells had been detached from the membrane. Tilted the        flask to ensure hose was at the edge of the flask.    -   12. Released all clamps leading to the 2 L TP and using the        GatheRex transfer the residual cell suspension into the 2 L TP        maintaining the tilted edge until all cells were collected.    -   13. Inspected membrane for adherent cells.    -   14. If necessary, back washed by releasing clamps on red line        and allowed some media to flow into the flask by gravity.    -   15. Closed the red line and triple heat seal.    -   16. Vigorously tapped flask to release cells.    -   17. Added cells to 2 L TP.    -   18. Heated seal the 2 L transfer pack leaving roughly the same        length of tubing as when dry weight was recorded.    -   19. Retained G-Rex 500MCS, it was reused in the split.    -   20. Recorded mass of transfer pack with cell suspension and        calculated the volume of cell suspension.    -   21. Determined cell suspension volume, including dry mass.    -   22. Sterile welded a 4″ transfer set to the cell suspension bag.    -   23. In the BSC mixed the cells gently and with 20 cc syringe        draw up 11 ml and aliquoted as shown in Table 14:

TABLE 14 Testing parameters. Test Sample volume Vessel Cell Count 2-2 mLsamples Cryovials and viability Mycoplasma   1 mL Cryovial stored at 4°C. until testing completed. Sterility   1 mL Inoculated 0.5 mL into oneeach anaerobic and aerobic culture bottles Flow 2-2 mL Unused cell count(Cryopreserved for future batch testing) Remainder of Discarded cells

-   -   24. Heat sealed. Closed the luer connection retaining the clamp    -   25. Labeled and placed the cell suspension in the incubator and        recorded time placed in the incubator.    -   26. Calculated new volume.    -   27. Recorded Volume in 2 L transfer pack based on volume of cell        suspension and volume removed for QC (11 mL).    -   28. Inoculated and ordered sterility testing.    -   29. Stored the mycoplasma sample at 4° C. in the pending rack        for mycoplasma testing.    -   30. Set aside until TIL was seeded.

Cell Count:

Performed single cell counts and recorded data and attach counting rawdata to batch record. Documented Dilution. Documented the Cellometercounting program. Verified the correct dilution was entered into theCellometer.

Method Continued:

-   -   31. Calculated the total number of flasks required for        subculture        -   *Re-used the original vessel and rounded fractions of            additional vessels up.

IL-2 Addition to CM

-   -   1. Placed 10 L bag of Aim V with Glutamax in the BSC.    -   2. Spiked the media bag with a 4″ plasma transfer set.    -   3. Attached an 18 G needle to a 10 mL syringe and draw 5 mL of        IL-2 into the syringe (final concentration is 3000 IU/ml).    -   4. Removed the needle and aseptically attach the syringe to the        plasma transfer set and dispensed IL-2 into the bag.    -   5. Flushed the line with air, draw up some media and dispense        into the bag. This insured all IL-2 is in the media.    -   6. Repeated for remaining bags of Aim V.

Prepare G-REX500MCS Flasks

-   -   1. Determined amount of CM4 to add to flasks. Recorded volume of        cells added per flask and volume of CM4 5000 mL-A.    -   2. Closed all clamps except the large filter line.    -   3. Sterile welded the inlet line of the Acacia pump to the 4″        plasma transfer set on the media bag containing CM4.    -   4. Sterile welded the outlet line of the pump to the G-Rex        500MCS via the red collection line.    -   5. Pump determined amount of CM4 into the G-Rex 500MCS using        lines on flask as guide.    -   6. Heated seal the G-Rex 500MCS red line.    -   7. Repeated steps 4-6 for each flask. Multiple flasks could be        filled at the same time using gravity fill or multiple pumps. A        “Y” connector could be welded to the outlet line of the pump and        the two arms welded to two G-Rex 500MCS flasks filling both at        the same time.    -   8. Placed flasks in a 37° C., 5% CO₂.

Seed Flasks With TIL

-   -   1. Closed all clamps on G-Rex 500MCS except large filter line    -   2. Sterile welded cell product bag to inlet line of the Acacia        pump.    -   3. Sterile welded the other end of the pump to the red line on        the G-Rex 500MCS.    -   4. Placed pump boot in pump.    -   5. Placed the cell product bag on analytical balance and        recorded time TIL added to G-REX flask.    -   6. Zeroed the balance.    -   7. Unclamped lines and pump required volume of cells into G-Rex        500MCS by weight assuming 1 g=1 mL.    -   8. Turned cell bag upside down and pump air to clear the line.        Heated seal red line of G-Rex 500MCS. Placed flask in incubator.    -   9. Sterile welded the outlet line of the pump to the next G-Rex        500MCS via the red collection line    -   10. Mixed cells well.    -   11. Repeated cell transfer for all flasks.    -   12. Placed flasks in a 37° C., 5% CO₂ and recorded time TIL        added to G REX flask.    -   13. Ordered testing for settle plates to the microbiology lab.    -   14. Recorded accession numbers.    -   15. Ordered testing for aerobic and anaerobic sterility.    -   16. Ensured delivery of plates and bottles to the microbiology        lab.

Cryopreservation of Flow or Excess Cells:

-   -   1. Calculated amount of freezing media required:        -   a. Target cell concentration was 1×10⁸/ml; record total            cells removed. Target cell concentration was 1×10⁸ cells/mL.            Calculated total volume of freezing media to add.    -   2. Prepared cryo preservation media and placed at 40° C. until        needed.    -   3. Spun down TIL at 400×g for 5 min at 20° C. with full brake        and full acceleration.    -   4. Aseptically aspirated supernatant.    -   5. Gently tapped bottom of tube to resuspend cells in remaining        fluid.    -   6. While gently tapping the tube slowly added prepared freezing        media.    -   7. Aliquoted into appropriate sized labelled cryo tubes.    -   8. Placed vial in a Mr. Frosty or equivalent and placed in a        −80° C. freezer.    -   9. Within 72 hours transferred to permanent storage location and        documented and recorded date and time placed in −80° C. freezer.

Example 8: Process 2A—Day 22

This example describes the detailed day 22 protocol for the 2A processdescribed in Examples 1 to 4.

Document Negative In-Process Sterility Results

Before beginning harvest, obtained the Day 16 preliminary sterilityresults from Microbiology lab. Contacted the Laboratory Director ordesignee for further instructions if the results were positive.

Clean Room Environmental Monitoring—Pre-Processing

-   -   1. Verified Particle Counts for 10 minutes before beginning        processing.    -   2. Biosafety Cabinets were cleaned with large saturated alcohol        wipes or alcohol spray.    -   3. Set up in-process surveillance plates and left in biosafety        cabinet for 1-2 hour during procedure.

Advanced Preparation

-   -   1. In BSC aseptically attached a Baxter extension set to a 10 L        labtainer bag or equivalent. Label LOVO filtrate bag.    -   2. Placed three 1 L bags of PlasmaLyte A in the BSC    -   3. Prepared pool and labeled the PlasmaLyte A bags with 1% HSA:        -   3.1. Closed all clamps on a 4S-4M60 Connector set and spiked            each of the PlasmaLyte bags.        -   3.2. Welded one of the male ends of the 4S-4M60 to the inlet            line of the Acacia pump boot.        -   3.3. Welded the outlet line of the pump boot to a 3 liter            collection bag. Closed all clamps on 3 L bag except the line            to pump.        -   3.4. Pumped the 3 liters of Plasmalyte into the 3 liter bag.            If necessary removed air from 3 L bag by reversing the pump.        -   3.5. Closed all clamps except line with female luer.        -   3.6. Using two 100 mL syringes and 16-18 G needles, load 120            mL of 25% HSA. Red capped syringes.        -   3.7. Attached one syringe to the female luer on the 3 liter            bag and transferred HSA to 3 L PlasmaLyte bag. Mix well.        -   3.8. Repeated with second syringe.        -   3.9. Mixed well.        -   3.10. Closed all clamps.        -   3.11. Using a 10 mL syringe, removed 5 mL of PlasmaLyte with            1% HSA from the needleless port on the 3 liter bag.        -   3.12. Capped syringe and kept in BSC for IL-2 dilution.        -   3.13. Closed all clamps.        -   3.14. Heated seal removing the female luer line from the            pump boot.        -   3.15. Labeled LOVO Wash buffer and date. Expired within 24            hrs at ambient temperature.

IL-2 Preparation

-   -   1. Dispensed Plasmalyte/1% HSA from 5 mL syringe into a labeled        50 ml sterile conical tube.    -   2. Added 0.05 mL IL-2 stock to the tube containing PlasmaLyte.    -   3. Labeled IL-2 6×10⁴    -   4. Capped label and store at 2-8° C. Record volumes.

Preparation of Cells

-   -   1. Closed all clamps on a 10 L Labtainerbag. At Attach Baxter        extension set to the 10 L bag via luer connection.    -   2. Removed the G-REX 500M flasks from the 37° C.    -   3. Sterile welded the red media removal line from the G-Rex        500MCS to the extension set on the 10 L bioprocess bag.    -   4. Sterile welded the clear cell removal line from the G-Rex        500MCS to a 3 L collection bag and labeled “pooled cell        suspension”.    -   5. Unclamped red line and 10 L bag.    -   6. Used the GatheRex pump, volume reduced the first flask.    -   Note: If an air bubble was detected then the pump could stop        prematurely. If full volume reduction was not complete        reactivated GatheRex pump.    -   7. Closed the clamp on the supernatant bag and red line.    -   8. Swirled the G-REX 500M flask until the TIL were completely        resuspended while avoiding splashing or foaming. Made sure all        cells have been dislodged from the membrane.    -   9. Opened clamps on clear line and 3 L cell bag.    -   10. Tilted the G-Rex flask such that the cell suspension was        pooled in the side of the flask where the collection straw was        located.    -   11. Started GatherRex to collect the cell suspension. Note: If        the cell collection straw was not at the junction of the wall        and bottom membrane, rapping the flask while tiled at a 45°        angle was usually sufficient to properly position the straw.    -   12. Ensured all cells had been removed from the flask.    -   13. If cells remained in the flask, added 100 mL of supernatant        back to the flask, swirled, and collected into the cell        suspension bag.    -   14. Closed clamp on the line to the cell collection bag.        Released clamps on GatheRex.    -   15. Heated seal clear line of G-Rex 500MCS.    -   16. Heated seal red line of G-Rex 500MCS.    -   17. Repeated steps 3-16 for additional flasks.    -   18. It was necessary to replace 10 L supernatant bag as needed        after every 2nd flask.    -   19. Multiple GatherRex could be used.    -   20. Documented number of G-Rex 500MCS processed.    -   21. Heated seal cell collection bag. Recorded number of G-REX        harvested.    -   22. With a marker made a mark ˜2″ from one of the female luer        connectors on a new 3 liter collection bag.    -   23. Heated seal and removed the female luer just below the mark.    -   24. Labeled as LOVO Source Bag    -   25. Recorded the dry weight.    -   26. Closed all clamps of a 170 μm blood filter.    -   27. Sterile welded the terminal end of the filter to the LOVO        source bag just below the mark.    -   28. Sterile welded a source line of the filter to the bag        containing the cell suspension.    -   29. Elevated the cell suspension by hanging cells on an IV pole        to initiate gravity-flow transfer of cells. (Note: Did not allow        the source bag to hang from the filtration apparatus.)    -   30. Opened all necessary clamps and allow TIL to drain from the        cell suspension bag through the filter and into the LOVO source        bag.    -   31. Once all cells were transferred to the LOVO source bag,        closed all clamps, heated seal just above the mark and detached        to remove filter.    -   32. Mixed bag well and using a two 3 mL syringe take 2        independent 2 mL samples from the syringe sample port for cell        counting and viability.    -   33. Weighed the bag and determined the difference between the        initial and final weight.    -   34. Recorded data and place in incubator, including dry mass.

Cell Count.

Performed a single cell count on each sample and recorded data andattach counting raw data to batch record. Documented the Cellometercounting program. Verified the correct dilution was entered into theCellometer. Determined total number of nucleated cells. Determinednumber of TNC to remove to retain=1.5×10¹¹ cells for LOVO processing.Place removed cell into appropriate size container for disposal.

LOVO Harvest

The 10 L Labtainer with Baxter extension set in Prior Preparation wasthe replacement filtrate bag welded to the LOVO kit later on. TurnedLOVO on and follow the screen displays.

Check Weigh Scales and Pressure Sensor

To access the Instrument Operation Profile:

-   -   1. Touched the information button.    -   2. Touched the instrument settings tab.    -   3. Touched the Instrument Operation Profile button.    -   4. The Instrument Operation Profile displayed.

Check the weigh scales

-   -   1. Made sure there was nothing hanging on any of the weigh        scales and reviewed the reading for each scale.    -   2. If any of the scales read outside of a range of 0+/−2 g,        performed weigh scale calibration as described in the Weigh        Scale Calibration Manual from the manufacturer.    -   3. If all scales were in tolerance with no weight hanging,        proceed to hang a 1-kg weight on each scale (#1-4) and reviewed        the reading.    -   4. If any of the scales read outside of a range of 1000+/−10 g        when a 1-kg weight was hanging, performed weigh scale        calibration as described in the LOVO Operator's Manual from the        manufacturer.

Check the Pressure Sensor

-   -   1. Reviewed the pressure sensor reading on the Instrument        Operation Profile Screen.    -   2. N/A: If the pressure sensor reading was outside 0+/−10 mmHg,        stored a new atmospheric pressure setting in Service Mode as        described in the LOVO Operator's Manual from the manufacturer.        -   a. Touched the check button on the Instrument Operation            Profile screen.        -   b. Touched the check button on the Instrument Settings tab.    -   3. If weigh scale calibration had been performed or a new        atmospheric pressure setting had been stored, repeated the        relevant sections.

To start the procedure, selected the “TIL G-Rex Harvest” protocol fromthe drop-down menu on the Protocol Selection Screen and press Start.

-   -   1. The Procedure Setup Screen displayed.    -   2. Touched the Solutions Information button.    -   3. The Solution 1 Screen displayed. Review the type of wash        buffer required for Solution 1. (Should read PlasmaLyte.)    -   4. Touched the Next button to advance to the Solution 2 Screen.        Reviewed the type of wash buffer required for Solution 2.        (Should read “NONE”, indicating that the protocol had been        configured to only use one type of wash buffer, which was        PlasmaLyte)    -   5. Touched the check button on the Solution 2 Information Screen        to return to the Procedure Setup screen.    -   6. Touched the Procedure Information Button.    -   7. The Procedure Information Screen displayed.    -   8. Touched the User ID entry field. A keypad will display.        Entered the initials of the performer and verifier. Touched the        button to accept the entry.    -   9. Touched the Source ID entry field. A keypad will display.        Entered the product lot #. Touched the button to accept the        entry.    -   10. Touched the Procedure ID entry field. A keypad will display.        Entered “TIL Harvest”. Touched the button to accept the entry.    -   11. If there are extra notes to record, touched the Procedure        Note entry field. A keypad displayed. Entered any notes. Touched        the button to accept the entry.    -   NOTE: The Procedure Note entry field is optional and can be left        blank.    -   12. Touched the check button on the Procedure Information Screen        to return to the Procedure Setup Screen.    -   13. Verified that a “check” displays in the Procedure        Information button. If no “check” displays, touched the        Procedure Information button again and ensured that the User ID,        Source ID, and Procedure ID fields all had entries.    -   14. Touched the Parameter Configuration Button.    -   15. The General Procedure Information Screen displayed.    -   16. Touched the Source Volume (mL) entry field. A numeric keypad        displayed. Entered the Calculated volume of cell suspension (mL)        from Table 1    -   17. Touched the button to accept the entry.    -   18. Touched the Source PCV (%) entry field. The TIL        (viable+dead) screen displays.    -   19. Touched the Cell Concentration entry field. A numeric keypad        displayed. Entered the Total Cellular concentration/mL from        Table 14 in the Source product in units of “×10⁶/mL”. The entry        could range from 00.0 to 99.9. Touched the button to accept the        entry and return to the General Procedure Information Screen.        NOTE: After the Cell Concentration was accepted, the Source PCV        (%) entry field on the General Procedure Information Screen        displayed the PCV % calculated by the LOVO, based on the Cell        Concentration entry made by the operator.    -   20. On the General Procedure screen, touched the Next button to        advance to screen 4 of 8, the Final Product Volume (Retentate        Volume) screen. Note: Screens 2 and 3 did not have any entry        fields for the operator to fill in.    -   21. The Final Product Volume (Retentate Volume) screen        displayed.    -   22. Using the Total nucleated cells (TNC) value from Table 15,        determined the final product target volume in the table below        (Table 16). Entered the Final Product Volume (mL) associated        with that Cell Range during LOVO Procedure setup.

TABLE 15 Determination of Final Product Target Volume. Final Product(Retentate) Volume to Cell Range Target (mL) 0 < Total (Viable + Dead)Cells ≤ 7.1E10 150 7.1E10 < Total (Viable + Dead) Cells ≤ 1.1E11 2001.1E11 < Total (Viable + Dead) Cells ≤ 1.5E11 250

TABLE 16 Product target volume. Total nucleated cells (TNC) × FinalProduct (Retentate) Target Volume 10⁶ (mL)  

-   -   23. To target the specified volume from Table 16 touched the        Final Product Volume (mL) entry field. A numeric keypad        displayed. Entered the desired Final Product Volume in unit of        mL. Touched the button to accept the entry.    -   24. Touched on the Final Product Volume (Retentate Volume)        screen to return to the Procedure Setup Screen. Note: Screens        5-8 did not have any entry fields for the operator to fill in.    -   25. Verified that a “Check” displays in the Parameter        Configuration button. If no “check” displays, touched the        Procedure Information button again and ensured that Source        Volume and Source PCV on page 1 have entries. Also ensured that        either the Target Minimum Final Product Volume checkbox was        checked OR the Final Product Volume (mL) field had an entry on        page 4.    -   26. Touched the Estimate Button at the top right corner of the        screen.    -   27. The Estimation Summary Screen displayed. Confirmed        sufficient and accurate values for Source and PlasmaLyte Wash        Buffer.    -   28. Loaded the disposable kit: Followed screen directions for        kit loading by selecting help button “(?)”.    -   29. Made a note of the volumes displayed for Filtrate and        Solution 1 (read PlasmaLyte)    -   30. Made a note of the volumes displayed for Filtrate and        Solution 1 (read PlasmaLyte).    -   31. For instructions on loading the disposable kit touched the        help button or followed instructions in operators manual for        detailed instructions.    -   32. When the standard LOVO disposable kit had been loaded,        touched the Next button. The Container Information and Location        Screen displays. Removed filtrate bag from scale #3.    -   33. For this protocol, the Filtrate container was New and Off        Scale    -   34. If the Filtrate container was already shown as New and        Off-Scale, no changes were made.    -   35. If the Filtrate container type was shown as Original,        touched the Original button to toggle to New.    -   36. If the Filtrate location was shown as On-Scale, touched the        On-Scale button to toggle to Off-Scale.    -   37. If the volume of Filtrate to be generated was ≤2500 mL, the        Filtrate Container    -   Location was shown as On-Scale For consistency among runs, the        Filtrate Container Location was changed to Off-Scale and        container type was “new”.    -   38. Touched the On-Scale button to toggle to Off-Scale. Attached        transfer set Use sterile welding technique to replace the LOVO        disposable kit Filtrate container with a 10-L bag. Opened the        weld.    -   39. Placed the Filtrate container on the benchtop. Did NOT hang        the Filtrate bag on weigh scale #3. Weigh scale #3 was empty        during the procedure.    -   40. Opened any plastic clamps on the tubing leading to the        Filtrate container. NOTE: If the tubing was removed from the F        clamp during welding, replaced in clamp.    -   41. Touched the Filtrate Container Capacity entry field. A        numeric keypad displayed. Entered the total new Filtrate        capacity (10,000 mL). Touched the “check” button to accept the        entry.    -   42. Used sterile welding technique to replace the LOVO        disposable kit Filtrate container with a 10-L bag. Opened the        weld. Note: If tubing was removed from the F clamp during        welding, replaced in clamp.    -   43. Placed the new Filtrate container on the benchtop. Did NOT        hang the Filtrate bag on weigh scale #3. Weigh scale #3 was        empty during the procedure    -   44. Opened any plastic clamps on the tubing leading to the        Filtrate container.    -   45. For the Retentate container, the screen displayed Original        and On-Scale.    -   46. No changes were made to the Retentate container.    -   47. When all changes were made to the Filtrate container and        appropriate information entered, touched the Next button.    -   48. The Disposable Kit Dry Checks overlay displays. Checked that        the kit had been loaded properly, then pressed the Yes button.    -   49. All LOVO mechanical clamps closed automatically and the        Checking Disposable Kit Installation screen displayed. The LOVO        went through a series of pressurizing steps to check the kit.    -   50. After the disposable kit check passed successfully, the        Connect Solutions screen displayed.    -   51. 3 L was the wash volume. Entered this value on screen.    -   52. Used sterile welding technique to attach the 3-L bag of        PlasmaLyte to the tubing passing through Clamp 1. Opened the        weld.    -   53. Hung the PlasmaLyte bag on an IV pole,    -   54. Opened any plastic clamps on the tubing leading to the        PlasmaLyte bag.    -   55. Verified that the Solution Volume entry is 3000 mL. This was        previously entered.    -   56. Touched the Next button. The Disposable Kit Prime overlay        displayed. Verified that the PlasmaLyte was attached and any        welds and plastic clamps on the tubing leading to the PlasmaLyte        were open, then touched the Yes button. NOTE: Because only one        type of wash buffer (PlasmaLyte) was used during the LOVO        procedure, no solution was attached to the tubing passing        through Clamp 2. The Roberts clamp on this tubing remained        closed during the procedure.    -   57. Disposable kit prime started and the Priming Disposable Kit        Screen displayed. Visually observed that PlasmaLyte moving        through the tubing connected to the bag of PlasmaL Lyte. If no        fluid was moving, pressed the Pause Button on the screen and        determined if a clamp or weld was still closed. Once the problem        had been solved, pressed the Resume button on the screen to        resume the Disposable Kit Prime.    -   58. When disposable kit prime finished successfully, the Connect        Source Screen displayed.    -   59. For this protocol, the Source container was New and        Off-Scale    -   60. If the Source container was already shown as New and        Off-Scale, no changes were made.    -   61. If the Source location was shown as On-Scale, touched the        On-Scale button to toggle to Off-Scale.    -   62. Touched the Source Capacity (mL) entry field. A numeric        keypad displayed. Enter the capacity of the container that held        the Source product. Touched the check button to accept the        entry. Note: The Source Capacity entry was used to make sure        that the Source bag was able to hold the additional solution        that was added to the bag during the Source Prime phase.    -   63. Used sterile welding technique to attach the Source        container to the tubing passing through Clamp S. Opened the        weld. Remove the tubing from the clamp as needed.    -   64. Made sure to replace source tubing into the S clamp.    -   65. Touched the Next button. The Source Prime overlay displayed.        Verified that the Source was attached to the disposable kit and        any welds and plastic clamps on the tubing leading to the Source        were open, then touched the Yes button.    -   66. Source prime started and the Priming Source Screen        displayed. Visually observed that PlasmaLyte was moving through        the tubing attached to the Source bag. If no fluid was moving,        pressed the Pause Button on the screen and determined if a clamp        or weld was still closed. Once the problem had been solved,        pressed the Resume button on the screen to resume the Source        Prime.    -   67. When Source prime finished successfully, the Start Procedure        Screen displayed.    -   68. Pressed the Start button. The “Pre-Wash Cycle 1” pause        screen appeared, with the instructions to “Coat IP, Mix Source”.    -   69. Pre-coated the IP bag.    -   70. Before pressing the Start button, removed the IP bag from        weigh scale #2 (could also remove tubing from the IP top port        tubing guide) and manually inverted it to allow the wash buffer        added during the disposable kit prime step to coat all interior        surfaces of the bag.    -   71. Re-hung the IP bag on weigh scale #2 (label on the bag faced        to the left). Replaced the top port tubing in the tubing guide,        if it was removed.    -   72. Mixed the Source bag.    -   73. Before pressing the Start button, removed the Source bag        from weigh scale #1 and inverted it several times to create a        homogeneous cell suspension.    -   74. Rehung the Source bag on weigh scale #1 or the IV pole. Made        sure the bag was not swinging.    -   75. Pressed the Start button.    -   76. The LOVO started processing fluid from the Source bag and        the Wash Cycle 1 Screen displayed.

During the LOVO procedure, the system automatically paused to allow theoperator to interact with different bags. Different screens displayedduring different pauses. Followed the corresponding instructions foreach screen.

Source Rinse Pause

After draining the Source bag, the LOVO added wash buffer to the Sourcebag to rinse the bag. After the configured volume of wash buffer hadbeen added to the Source bag, the LOVO paused automatically anddisplayed the Source Rinse Paused Screen.

When the Source Rinse Paused Screen displayed, the operator:

-   -   1. Removed the Source bag from weigh scale #1.    -   2. Inverted the Source bag several times to allow the wash        buffer to touch the entire bag interior.    -   3. Re-hung the Source bag on weigh scale #1. Made sure the        Source bag is not swinging on weigh scale #1.    -   4. Pressed the Resume button.

The LOVO processed the rinse fluid from the Source bag, then continuedwith the automated procedure.

Mix IP Bag Pause

To prepare cells for another pass through the spinner, the IP bag wasdiluted with wash buffer. After adding the wash buffer to the IP bag,the LOVO paused automatically and displayed the “Mix IP bag” PauseScreen.

When the “Mix IP bag” Pause Screen displayed, the operator:

-   -   1. Removed the IP bag from weigh scale #2. Could also remove the        tubing from the IP top port tubing guide.    -   2. Inverted the IP bag several times to thoroughly mix the cell        suspension.    -   3. Re-hung the IP bag on weigh scale #2. Also replaced the IP        top port tubing in the tubing guide, if it was removed. Made        sure the IP bag was not swinging on weigh scale #2.    -   4. Pressed the Resume button. The LOVO began processing fluid        from the IP bag.

Massage IP Corners Pause

During the final wash cycle of the LOVO procedure, cells were pumpedfrom the IP bag, through the spinner, and to the Retentate (FinalProduct) bag. When the IP bag was empty, 10 mL of wash buffers was addedto the bottom port of the IP bag to rinse the bag. After adding therinse fluid, the LOVO paused automatically and displayed the “Massage IPcorners” Pause Screen.

When the “Massage IP corners” Pause Screen displayed, the operator:

-   -   1. Did NOT remove the IP bag from weigh scale #2.    -   2. With the IP bag still hanging on weigh scale #2, massaged the        corners of the bag to bring any residual cells into suspension.    -   3. Made sure the IP bag was not swinging on weigh scale #2.    -   4. Pressed the Resume button.    -   5. The LOVO began pumping out the rinse fluid from the IP bag.

At the end of the LOVO procedure, the Remove Products Screen displayed.When this screen displayed, all bags on the LOVO kit could bemanipulated.

Note: Did not Touch any Bags Until the Remove Products Screen Displays.

Placed a hemostat on the tubing very close to the port on the Retentatebag to keep the cell suspension from settling into the tubing and tripleheat sealed below the hemostat.

Removed the retentate bag by breaking the middle seal and transferred tothe BSC.

Followed the instructions on the Remove Products Screen

Touched the Next button. All LOVO mechanical clamps opened and theRemove Kit Screen displayed.

Followed the instructions on the Remove Kit screen. When completedproceeded.

Touched the Next button. All LOVO mechanical clamps closed and theResults Summary Screen displayed. Recorded the data from the resultssummary screen in Table 17. Closed all pumps and filtered support.

TABLE 17 LOVO results summary table. Elapsed Elapsed Source ProcessingProcessing Time Time Source Retentate Filtrate Solution 1 (paren-(paren- Pause Volume Volume Volume Volume theses #) theses #) Time (mL)(mL) (mL) (mL) A. B. C. D. E. F. G.

Touched the Next button. The Protocol Selection Screen displayed.

LOVO Shutdown Procedure

-   -   1. Ensured all clamps were closed and filter support is in the        upright position.    -   2. Touched the STOP button on the front of the LOVO.    -   3. The STOP Button Decision Overlay displayed.    -   4. The Shutdown Confirmation Overlay displayed.    -   5. Touched the Yes button. The Shutting Down Screen displayed.    -   6. After a few seconds, the Power Off Screen displayed. When        this screen displayed, turned off the LOVO using the switch on        the back left of the instrument.

Recorded final formulated product volume in a table.

Calculate amount of IL-2 required from final product table. See FIG.129.

Determined the number of Cryobags and Retain Volume Marked on the Targetvolume and retain table below the number of cryopreservation bags andvolume of retention sample for product.

Targeted volume/bag calculation: (Final formulated volume −volumeadjustment due to not getting 100% recovery=10 mL)/# bags.

Prepared cells with 1:1 (vol:vol) CS10 (CryoStor 10, BioLife Solutions)and IL-2.

-   -   1. Assemble Connect apparatus        -   1.1. Sterile welded the CS750 cryobags to the CC2 Cell            Connect apparatus replacing one of the distal male luer ends            for each bag.        -   1.2. Retained the clamps in the closed position.        -   1.3. Labeled the bags 1-4.    -   2. Prepared cells with IL-2 and connected apparatus.        -   2.1. In BSC spike the cell product bag with a 4″ plasma            transfer set with female luer connector. Be sure the clamp            was closed on the transfer set.        -   2.2. With an appropriate size syringe drew up the volume of            IL-2 working dilution determined from the Final Product            Table.        -   2.3. Dispensed into LOVO product.        -   2.4. Sterile welded LOVO product bag to CC2 single spike            line removing the spike.        -   2.5. Placed cells and apparatus in transport bag and place            at 2-8° C. for ≤15 min.    -   3. Addition of CS10        -   3.1. In BSC attached 3 way stopcock to male luer on bag of            cold CS10.        -   3.2. Attached appropriate size syringe to female luer of            stopcock.        -   3.3. Unclamped bag and drew up the amount of CS10 determined            in the “Final Formulated Product Volume” table.        -   3.4. Removed syringe and red capped.        -   3.5. Repeated if multiple syringes were required.        -   3.6. Removed cell/CC2 apparatus from 2-8° C. refrigerator            and placed in BSC.        -   3.7. Attached first syringe containing CS10 to middle luer            of stopcock. Turned stopcock so line to CS750 bags is in            “OFF” position.        -   3.8. Slowly and with gentle mixing, added CS10 (1:1,            vol:vol) to cells.        -   3.9. Repeated for additional syringes of CS10.

Addition of Formulated Cell Product into Cryobags

-   -   1. Replaced syringe with appropriate size syringe for volume of        cells to be placed in each cryo bag.    -   2. Mixed cell product.    -   3. Opened the clamp leading to the cell product bag and drew up        appropriate volume    -   4. Turned stopcock so cell product bag is in “OFF” position and        dispensed the contents of the syringe into cryobag #1. Cleared        the line with air from syringe.

Record final product volume

-   -   1. Using needless port and appropriate size syringe, drew up        amount of retain determined previously.    -   2. Place retained in 50 mL conical tube labelled “Retain”    -   3. Using the syringe attached to the harness removed all air        from bag drawing up cells to about 1″ past bag into tubing.        Clamped and heat sealed. Placed at 2-8° C.    -   4. Turned stopcock so cryo bags were in the “OFF” position    -   5. Mixed cells in cell product bag and repeat steps 3-8 for        remaining CS750 bags using a new syringe on the stopcock and new        syringe to obtain cell retain.    -   6. Retained should be set aside for processing once product was        in CRF.

Controlled-Rate Freezer (CRF) Procedure (See Also Example 9)

-   -   1. Turned on the CRF (CryoMed Controlled Rate Freezer,        Model 7454) and associated laptop computer.    -   2. Logged onto the computer using account and password    -   3. Opened Controlled Rate Freezer icon located on the desktop.    -   4. Clicked the Run button on the Main screen.    -   5. Clicked Open Profile, Click Open.    -   6. Entered the Run File Name followed by the date in this        format: runMMDDYYYY.    -   7. Entered the Data Tag as the date with no dashes as MMDDYYY.    -   8. Closed door to the CRF.    -   9. Clicked Start Run.    -   10. Selected COM 6 on the pull down menu.    -   11. Clicked Ok. Waited about 30 seconds.    -   12. When “Profile Download,” pops up, Clicked OK. Clicked Save.        (See Example 9 for controlled-rate freezing profile details.)    -   13. Waited to press green button until the samples were in the        CRF. The freezer was held at 4° C. until ready to add them.    -   14. Added samples to CRF.    -   15. Waited until CRF returns to 4° C. Once temperature was        reached, clicked the green continue button. This initiated        program to go to next step in program.    -   16. Performed a visual inspection of the cryobags for the        following (Note: did not inspect for over or underfill):        container integrity, port integrity, seal integrity, presence of        cell clumps, and presence of particles.    -   17. Placed approved hang tag labels on each bag.    -   18. Verified final product label including: Lot number, product        name, manufacturer date, product volume, other additives,        storage temperature, and expiration.    -   19. Placed each cryobag (with hangtag) into an over-bag.    -   20. Heat sealed.    -   21. Placed in a cold cassette.    -   22. Repeated for each bag.    -   23. Placed the labeled cryobags into preconditioned cassettes        and transferred to the CRF.    -   24. Evenly distributed the cassettes in the rack in the CRF.    -   25. Applied ribbon thermocouple to the center cassette, or place        dummy bag in center position.    -   26. Closed the door to the CRF.    -   27. Once the chamber temperature reached 4° C.+/−1.5° C., Press        Run on the PC Interface software.    -   28. Recorded the time and the chamber temperature that the        product is transferred to the CRF.

Processing of Quality Control Sample

-   -   1. Aseptically transferred the following materials to the BSC,        as needed, and labeled according to the table below:    -   2. Used a new pipette for pipette the following:        -   QC and Retention Table    -   3. Delivered to QC: 1—Cell Count tube, 1—Endotoxin tube,        1—Mycoplasma tube, 1—Gram stain tube, 1 tube restimulation tube,        and 1—flow tube to QC for immediate testing. The remaining        duplicate tubes were placed in the controlled rate freezer.    -   4. Contacted the QC supervisor notifying of required testing.    -   5. See Table 18 for testing and storage instructions.

TABLE 18 Testing and storage instructions. Test Vessel Cell Count andCryovials. viability Mycoplasma Cryovial stored at 4° C. until testingcompleted. Sterility Inoculate 0.5 mL into an anaerobic and 0.5 mL intoan aerobic culture bottle. Gram Stain Cryovial stored at 4° C. untiltesting completed. Endotoxin Cryovial stored at 4° C. until testingcompleted. Flow Cryovial stored at 4° C. until testing completed. PostCryopreserve for future testing: Consist of 5 satellite vial,Formulation 1-Cell Count tube, 1-Endotoxin tube, 1-Mycoplasma Retentiontube, 1-Gram stain tube, and 1-flow tube to QC for immediate testing.Restimulation Sample is delivered at room temperature and assay must bestarted within 30 minutes of cell count results.

Cell Count

Performed a single cell count on each sample and recorded data andattached counting raw data to batch record. Document the Cellometercounting program. Verified the correct dilution was entered into theCellometer.

Cryopreservation of Post Formulation Retention Cells:

-   -   1. Placed vial in CRF.    -   2. Moved to storage location after completion of freeze and        recorded date and time placed in CFR. Recorded date and time        moved to LN2.

Microbiology Testing

-   -   1. Ordered testing for settle plates to the microbiology lab.    -   2. Recorded accession numbers.    -   3. Ordered testing for aerobic and anaerobic sterility.    -   4. Ensured delivery of plates and bottles to the microbiology        lab.

Post-Cryopreservation of Cell Product Bags

-   -   1. Stopped the freezer after the completion of the run. Run        could be stopped by clicking on the Stop button or pressing the        Back key on the freezer keypad.    -   2. Removed cryobags from cassette    -   3. Transferred cassettes to vapor phase LN2.    -   4. Recorded storage location.    -   5. Entered any additional comments when the text entry window        opens again. This window appeared regardless of the Run stop        method.    -   6. Printed the profile report and attached to the batch record        labeled with the lot number for the run.    -   7. Terminated Warm Mode and closed the Run screen with Exit        button.

Example 9: Cryopreservation Process

This example describes the cryopreservation process method for TILsprepared with the abbreviated, closed procedure described above inExample 8 using the CryoMed Controlled Rate Freezer, Model 7454 (ThermoScientific).

The equipment used, in addition to that described in Example 9, is asfollows: aluminum cassette holder rack (compatible with CS750 freezerbags), cryostorage cassettes for 750 mL bags, low pressure (22 psi)liquid nitrogen tank, refrigerator, thermocouple sensor (ribbon type forbags), and CryoStore CS750 Freezing bags (OriGen Scientific).

The freezing process provides for a 0.5° C. rate from nucleation to −20°C. and 1° C. per minute cooling rate to −80° C. end temperature. Theprogram parameters are as follows: Step 1—wait at 4° C.; Step 2: 1.0°C./min (sample temperature) to −4° C.; Step 3: 20.0° C./min (chambertemperature) to −45° C.; Step 4: 10.0° C./min (chamber temperature) to−10.0° C.; Step 5: 0.5° C./min (chamber temperature) to −20° C.; andStep 6: 1.0° C./min (sample temperature) to −80° C.

A depiction of the procedure of this example in conjunction with theprocess of Examples 1 to 8 is shown in FIG. 11.

Example 10: Characterization of Process 2A TILs

This example describes the characterization of TILs prepared with theabbreviated, closed procedure described above. In summary, theabbreviated, closed procedure (process 2A, described in Examples 1 to 9)had the advantages over prior TIL manufacturing processes given in Table19. Advantages for the Pre-REP can include: increased tumor fragmentsper flask, shortened culture time, reduced number of steps, and/or beingamenable to closed system. Advantages for the Pre-REP to REP transitioncan include: shortened pre-REP-to-REP process, reduced number of steps,eliminated phenotyping selection, and/or amenable to closed system.Advantages for the REP can include: reduced number of steps, shorter REPduration, closed system transfer of TIL between flasks, and/or closedsystem media exchanges. Advantages for the Harvest can include: reducednumber of steps, automated cell washing, closed system, and reduced lossof product during wash. Advantages for the final formulation and/orproduct can include shipping flexibility.

TABLE 19 Comparison of exemplary process 1C and an embodiment of process2A. Process Step Process 1C - Embodiment Process 2A - Embodiment Pre-REP4 fragments per 10 G-REX-10 flasks 40 fragments per 1 G-REX-100M flask11-21 day duration 11 day duration Pre-REP to Pre-REP TIL are frozenuntil Pre-REP TIL directly move to REP REP phenotyped for selection thenthawed on day 11 Transition to proceed to the REP (~day 30) REPrequires >40 × 10⁶ TIL REP requires 25-200 × 10⁶ TIL REP 6 G-REX-100Mflasks on REP day 0 1 G-REX-500M flask on day 11 5 × 10⁶ TIL and 5 × 10⁸PBMC feeders 25-200 × 10⁶ TIL and 5 × 10⁹ PBMC per flask on REP day 0feeders on day 11 Split to 18-36 flasks on REP day 7 Split to ≤6G-REX-500M flasks on day 16 14 day duration 11 day duration Harvest TILharvested via centrifugation TIL harvested via LOVO automated cellwashing system′ Final Fresh product in Hypothermosol Cryopreservedproduct in Formulation PlasmaLyte-A + 1% HSA and CS10 stored in LN₂Single infusion bag Multiple aliquots Limited shipping stability Longershipping stability Overall 43-55 days 22 days Estimated Process Time

A total of 9 experiments were performed using TILs derived from 9 tumorsdescribed in Table 20. All the data shown here was measured from thawedfrozen TIL product from process 1C and an embodiment of process 2A.

TABLE 20 Description of Tumor Donors, Processing Dates and ProcessingLocations. Tumor ID Tissue type Source Tissue M1061 Melanoma MT groupPrimary - left lateral foot M1062 Melanoma Moffitt N/A M1063 Melanoma MTgroup Metastatic C - right groin M1064 Melanoma MT group Metastatic C -left ankle M1065 Melanoma Bio Metastatic-Axillary lymph node OptionsEP11001 ER+PR+ MT group Primary - left breast invasive ductal carcinomaM1056* Melanoma Moffitt N/A M1058* Melanoma MT group Metastatic - StageIIB Right scalp M1023* Melanoma Atlantic Primary - Right axilla Health

The procedures described herein for process 2A were used to produce theTILs for characterization in this example. Briefly, for the REP, on Day11, one G-REX-500M flask containing 5 L of CM2 supplemented with 3000IU/ml rhil-2, 30 ng/mL anti-CD3 (Clone OKT3) and 5×10⁹ irradiatedallogeneic feeder PBMC cells was prepared. TILs harvested from thepre-REP G-REX-100M flask after volume reduction were counted and seededinto the G-REX-500M flask at a density that ranged between 5×10⁶ and200×10⁶ cells. The flask was then placed in a humidified 37° C., 5% CO₂tissue culture incubator for five days. On Day 16, volume of theG-REX-500M flask was reduced, TILs were counted and their viabilitydetermined. At this point, the TIL were expanded into multipleG-REX-500M flasks (up to a maximum of six flasks), each with a seedingdensity of 1×10⁹ TILs/flask. All flasks were then placed in humidified37° C., 5% CO₂ tissue culture incubators for an additional six days. OnDay 22, the day of harvest, each flask was volume reduced by 90%, thecells were pooled together and filtered through a 170 μm blood filter,and then collected into a 3 L Origin EV3000 bag or equivalent inpreparation for automated washing using the LOVO. TILs were washed usingthe LOVO automated cell processing system which replaced 99.99% of cellculture media with a wash buffer consisting of PlasmaLyte-A supplementedwith 1% HSA. The LOVO operates using spinning filtration membranetechnology that recovers over 92% of the TIL while virtually eliminatingresidual tissue culture components, including serum, growth factors, andcytokines, as well as other debris and particulates. After completion ofthe wash, a cell count was performed to determine the expansion of theTILs and their viability upon harvest. CS10 was added to the washed TILat a 1:1 volume:volume ratio to achieve the Process 2A finalformulation. The final formulated product was aliquoted into cryostoragebags, sealed, and placed in pre-cooled aluminum cassettes. Cryostoragebags containing TIL were then frozen using a CryoMed Controlled RateFreezer (ThermoFisher Scientific, Waltham, Mass.) according to theprocedures described herein, including in Example 9.

Cell counts and percentage viability for the nine runs were compared inFIGS. 12 and 13.

The cell surface markers shown in the following results were analyzedusing flow cytometry (Canto II flow cytometer, Becton, Dickinson, andCo., Franklin Lakes, N.J., USA) using suitable commercially-availablereagents. Results for markers of interest are shown in FIG. 14 throughFIG. 23.

Diverse methods have been used to measure the length of telomeres ingenomic DNA and cytological preparations. The telomere restrictionfragment (TRF) analysis is the gold standard to measure telomere length(de Lange et al., 1990). However, the major limitation of TRF is therequirement of a large amount of DNA (1.5 μs). Here, two widely usedtechniques for the measurement of telomere lengths were applied, namelyfluorescence in situ hybridization (FISH) and quantitative PCR.

Flow-FISH was performed using the Dako kit (K532711-8 RUO Code K5327Telomere PNA Kit/FITC for Flow Cytometry, PNA FISH Kit/FITC. Flow, 20tests) and the manufacturer's instructions were followed to measureaverage length of telomere repeat. Briefly, the cells were surface wasstained with CD3 APC for 20 minutes at 4° C., followed by GAM Alexa 546for 20 minutes. The antigen-antibody complex was then cross-linked with2 mM BS3 (Fisher Scientific) chemical cross-linker. PNA-telomere probebinding in a standard population of T-cells with long telomeres, Jurkat1301 T leukemia cell line (1301 cells) was used as an internal referencestandard in each assay. Individual TILs were counted following antibodyincubation and mixed with 1301 cells (ATCC) at a 1:1 cell ratio. 5×10⁵TILs were mixed with 5×10⁵ 1301 cells. In situ hybridization wasperformed in hybridization solution (70% formamide, 1% BSA, 20 mM TrispH 7.0) in duplicate and in the presence and absence of aFITC-conjugated Telomere PNA probe (Panagene),FITC-00-CCC-TAA-CCC-TAA-CCC-TAA, complementary to the telomere repeatsequence at a final concentration of 60 nM. After addition of theTelomere PNA probe, cells were incubated for 10 minutes at 81° C. in ashaking water bath. The cells were then placed in the dark at roomtemperature overnight. The next morning, excess telomere probe wasremoved by washing 2 times with PBS pre-warmed to 40° C. Following thewashes, DAPI (Invitrogen, Carlsbad, Calif.) was added at a finalconcentration of 75 ng/mL. DNA staining with DAPI was used to gate cellsin the G0/G1 population. Sample analysis was performed using our flowcytometer (BD Canto II, Mountain View, Calif.). Telomere fluorescence ofthe test sample was expressed as a percentage of the fluorescence (fl)of the 1301 cells per the following formula: Relative telomerelength=[(mean FITC fl test cells w/probe-mean FITC fl test cells w/oprobe)×DNA index 1301 cells×100]/[(mean FITC fl 1301 cells w/probe−meanFITC fl 1301 cells w/o probe)×DNA index test cell.

Real time qPCR was also used to measure relative telomere length(Nucleic Acids Res. 2002 May 15; 30(10): e47., 20, Leukemia, 2013, 27,897-906). Briefly, the telomere repeat copy number to single gene copynumber (T/S) ratio was determined using an BioRad PCR thermal cycler(Hercules, Calif.) in a 96-well format. Ten ng of genomic DNA was usedfor either the telomere or hemoglobin (hgb) PCR reaction and the primersused were as follows: Tel-1b primer (CGG TTT GTT TGG GTT TGG GTT TGG GTTTGG GTT TGG GTT), Tel-2b primer (GGC TTG CCT TAC CCT TAC CCT TAC CCT TACCCT TAC CCT), hgb1 primer (GCTTCTGACACAACTGTGTTCACTAGC), and hgb2 primer(CACCAACTTCATCCACGTTCACC). All samples were analyzed by both thetelomere and hemoglobin reactions, and the analysis was performed intriplicate on the same plate. In addition to the test samples, each96-well plate contained a five-point standard curve from 0.08 ng to 250ng using genomic DNA isolated from 1301 cell line. The T/S ratio (−dCt)for each sample was calculated by subtracting the median hemoglobinthreshold cycle (Ct) value from the median telomere Ct value. Therelative T/S ratio (−ddCt) was determined by subtracting the T/S ratioof the 10.0 ng standard curve point from the T/S ratio of each unknownsample.

Flow-FISH results are shown in FIGS. 24 and 25, and no significantdifferences were observed between process 1C and process 2A, suggestingthat the surprising properties of the TILs produced by process 2A werenot predictable from the age of the TILs alone.

In conclusion, process 2A produced a potent TIL product with a “young”phenotype as defined by high levels of co-stimulatory molecules, lowlevels of exhaustion markers, and an increased capability to secretecytokine upon reactivation. The abbreviated 22 day expansion platformallows for the rapid generation of clinical scale doses of TILs forpatients in urgent need of therapy. The cryopreserved drug productintroduces critical logistical efficiencies allowing rapid manufactureand flexibility in distribution. This expansion method overcomestraditional barriers to the wider application of TIL therapy.

Example 11: Use of IL-2, IL-15, and IL-21 Cytokine Cocktail

This example describes the use of IL-2, IL-15, and IL-21 cytokines,which serve as additional T cell growth factors, in combination with theTIL process of Examples 1 to 10.

Using the process of Examples 1 to 10, TILs were grown from colorectal,melanoma, cervical, triple negative breast, lung and renal tumors inpresence of IL-2 in one arm of the experiment and, in place of IL-2, acombination of IL-2, IL-15, and IL-21 in another arm at the initiationof culture. At the completion of the pre-REP, cultures were assessed forexpansion, phenotype, function (CD107a+ and IFN-γ) and TCR Vβrepertoire. IL-15 and IL-21 are described elsewhere herein and inGruijl, et al., IL-21 promotes the expansion of CD27+CD28+ tumorinfiltrating lymphocytes with high cytotoxic potential and lowcollateral expansion of regulatory T cells, Santegoets, S. L, TranslMed., 2013, 11:37(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3626797/).

The results showed that enhanced TIL expansion (>20%), in both CD4⁺ andCD8⁺ cells in the IL-2, IL-15, and IL-21 treated conditions wereobserved in multiple histologies relative to the IL-2 only conditions.There was a skewing towards a predominantly CD8⁺ population with askewed TCR Vβ repertoire in the TILs obtained from the IL-2, IL-15, andIL-21 treated cultures relative to the IL-2 only cultures. IFN-γ andCD107a were elevated in the IL-2, IL-15, and IL-21 treated TILs, incomparison to TILs treated only IL-2.

Example 12: Phase 2, Multicenter, Three-Cohort Study in Melanoma

This Phase 2, multicenter, three-cohort study is designed to assess thesafety and efficacy of a TIL therapy manufactured according to process1C (as described herein) in patient with metastatic melanoma. Cohortsone and two will enroll up to 30 patients each and cohort three is are-treatment cohort for a second TIL infusion in up to ten patients. Thefirst two cohorts are evaluating two different manufacturing processes:processes 1C and an embodiment of process 2A (described in Examples 1 to10, respectively. Patients in cohort one receive fresh,non-cryopreserved TIL and cohort two patients receive productmanufactured through the process described in Examples 1 to 10, yieldinga cryopreserved product. The study design is shown in FIG. 26. The studyis a Phase 2, multicenter, three cohort study to assess the safety andefficacy of autologous TILs for treatment of subpopulations of patientswith metastatic melanoma. Key inclusion criteria include: measurablemetastatic melanoma and ≥1 lesion resectable for TIL generation; atleast one prior line of systemic therapy; age ≥18; and ECOG performancestatus of 0-1. Treatment cohorts include non-cryopreserved TIL product(prepared using process 1C), cryopreserved TIL product (prepared usingan embodiment of process 2A), and retreatment with TIL product forpatients without response or who progress after initial response. Theprimary endpoint is safety and the secondary endpoint is efficacy,defined as objective response rate (ORR), complete remission rate (CRR),progression free survival (PFS), duration of response (DOR), and overallsurvival (OS).

Example 13: Qualifying Individual Lots of Gamma-Irradiated PeripheralMononuclear Cells

This Example describes a novel abbreviated procedure for qualifyingindividual lots of gamma-irradiated peripheral mononuclear cells (PBMCs,also known as MNC) for use as allogeneic feeder cells in the exemplarymethods described herein.

Each irradiated MNC feeder lot was prepared from an individual donor.Each lot or donor was screened individually for its ability to expandTIL in the REP in the presence of purified anti-CD3 (clone OKT3)antibody and interleukin-2 (IL-2). In addition, each lot of feeder cellswas tested without the addition of TIL to verify that the received doseof gamma radiation was sufficient to render them replicationincompetent.

Definitions/Abbreviations

-   BSC—Biological Safety Cabinet-   CD3—Cluster of Differentiation 3; surface marker protein    T-lymphocytes-   CF—Centrifugal-   CM2—Complete Medium for TIL #-   CMO—Contract Manufacturing Organization-   CO₂—Carbon Dioxide-   EtOH—Ethyl Alcohol-   GMP—Good Manufacturing Practice-   IL-2—Interleukin 2-   IU—International Units-   LN2—Liquid Nitrogen-   mini-REP—Mini-Rapid Expansion Protocol-   ml—Milliliter-   MNC—Mononuclear Cells-   NA—Not Applicable-   OKT3—MACS GMP CD3 pure (clone OKT3) antibody-   PPE—Personal Protective Equipment-   Pre-REP—Before Rapid Expansion Protocol-   QS—Quantum Satis; fill to this quantity-   REP—Rapid Expansion Protocol-   TIL—Tumor Infiltrating Lymphocytes-   T25—25 cm² tissue culture flask-   μg—Micrograms-   μL—Microliter    Procedure    Background    -   7.1.1 Gamma-irradiated, growth-arrested MNC feeder cells were        required for REP of TIL. Membrane receptors on the feeder MNCs        bind to anti-CD3 (clone OKT3) antibody and crosslink to TIL in        the REP flask, stimulating the TIL to expand. Feeder lots were        prepared from the leukapheresis of whole blood taken from        individual donors. The leukapheresis product was subjected to        centrifugation over Ficoll-Hypaque, washed, irradiated, and        cryopreserved under GMP conditions.    -   7.1.2 It is important that patients who received TIL therapy not        be infused with viable feeder cells as this can result in        Graft-Versus-Host Disease (GVHD). Feeder cells are therefore        growth-arrested by dosing the cells with gamma-irradiation,        resulting in double strand DNA breaks and the loss of cell        viability of the MNC cells upon reculture.        Evaluation Criteria and Experimental Set-Up

Feeder lots were evaluated on two criteria: 1) their ability to expandTIL in co-culture >100-fold and 2) their replication incompetency.

-   -   7.2.2 Feeder lots were tested in mini-REP format utilizing two        primary pre-REP TIL lines grown in upright T25 tissue culture        flasks.    -   7.2.3 Feeder lots were tested against two distinct TIL lines, as        each TIL line is unique in its ability to proliferate in        response to activation in a REP.    -   7.2.4 As a control, a lot of irradiated MNC feeder cells which        has historically been shown to meet the criteria of 7.2.1 was        run alongside the test lots.    -   7.2.5 To ensure that all lots tested in a single experiment        receive equivalent testing, sufficient stocks of the same        pre-REP TIL lines were available to test all conditions and all        feeder lots.    -   7.2.6 For each lot of feeder cells tested, there was a total of        six T25 flasks:        -   7.2.6.1 Pre-REP TIL line #1 (2 flasks)        -   7.2.6.2 Pre-REP TIL line #2 (2 flasks)        -   7.2.6.3 Feeder control (2 flasks)        -   NOTE: Flasks containing TIL lines #1 and #2 evaluated the            ability of the feeder lot to expand TIL. The feeder control            flasks evaluated the replication incompetence of the feeder            lot.

Experimental Protocol

-   -   7.3.1 Day-2/3, Thaw of TIL lines        -   7.3.1.1 Prepared CM2 medium.        -   7.3.1.2 Warmed CM2 in 37° C. water bath.        -   7.3.1.3 Prepared 40 ml of CM2 supplemented with 3000 IU/ml            IL-2. Keep warm until use.        -   7.3.1.4 Placed 20 ml of pre-warmed CM2 without IL-2 into            each of two 50 ml conical tubes labeled with names of the            TIL lines used.        -   7.3.1.5 Removed the two designated pre-REP TIL lines from            LN2 storage and transferred the vials to the tissue culture            room.        -   7.3.1.6 Recorded TIL line identification.        -   7.3.1.7 Thawed vials by placing them inside a sealed zipper            storage bag in a 37° C. water bath until a small amount of            ice remains.        -   7.3.1.8 Sprayed or wiped thawed vials with 70% ethanol and            transfer vials to BSC.        -   7.3.1.9 Using a sterile transfer pipet, immediately            transferred the contents of vial into the 20 ml of CM2 in            the prepared, labeled 50 ml conical tube.        -   7.3.1.10 QS to 40 ml using CM2 without IL-2 to wash cells.        -   7.3.1.11 Centrifuged at 400×CF for 5 minutes.        -   7.3.1.12 Aspirated the supernatant and resuspend in 5 ml            warm CM2 supplemented with 3000 IU/ml IL-2.        -   7.3.1.13 Removed small aliquot (20 μl) in duplicate for cell            counting using an automated cell counter. Record the counts.        -   7.3.1.14 While counting, placed the 50 ml conical tube with            TIL cells into a humidified 37° C., 5% CO₂ incubator, with            the cap loosened to allow for gas exchange.        -   7.3.1.15 Determined cell concentration and dilute TIL to            1×10⁶ cells/ml in CM2 supplemented with IL-2 at 3000 IU/ml.        -   7.3.1.16 Cultured in 2 ml/well of a 24-well tissue culture            plate in as many wells as needed in a humidified 37° C.            incubator until Day 0 of the mini-REP.        -   7.3.1.17 Cultured the different TIL lines in separate            24-well tissue culture plates to avoid confusion and            potential cross-contamination.    -   7.3.2 Day 0, initiate Mini-REP        -   7.3.2.1 Prepared enough CM2 medium for the number of feeder            lots to be tested. (e.g., for testing 4 feeder lots at one            time, prepared 800 ml of CM2 medium).        -   7.3.2.2 Aliquoted a portion of the CM2 prepared in 7.3.2.1            and supplement it with 3000 IU/ml IL-2 for the culturing of            the cells. (e.g., for testing 4 feeder lots at one time,            prepare 500 ml of CM2 medium with 3000 IU/ml IL-2).        -   7.3.2.3 The remainder of the CM2 with no IL-2 will be used            for washing of cells as described below.        -   7.3.2.4 Working with each TIL line separately to prevent            cross-contamination, removed the 24-well plate with TIL            culture from the incubator and transferred to the BSC.        -   7.3.2.5 Using a sterile transfer pipet or 100-1000 W            Pipettor and tip, removed about 1 ml of medium from each            well of TIL to be used and place in an unused well of the            24-well tissue culture plate. This was used for washing            wells.        -   7.3.2.6 Using a fresh sterile transfer pipet or 100-1000 W            Pipettor and tip, mixed remaining medium with TIL in wells            to resuspend the cells and then transferred the cell            suspension to a 50 ml conical tube labeled with the TIL name            and recorded the volume.        -   7.3.2.7 Washed the wells with the reserved media and            transferred that volume to the same 50 ml conical tube.        -   7.3.2.8 Spun the cells at 400×CF to collect the cell pellet.        -   7.3.2.9 Aspirated off the media supernatant and resuspend            the cell pellet in 2-5 ml of CM2 medium containing 3000            IU/ml IL-2, volume to be used based on the number of wells            harvested and the size of the pellet—volume should be            sufficient to ensure a concentration of >1.3×10⁶ cells/ml.        -   7.3.2.10 Using a serological pipet, mixed the cell            suspension thoroughly and recorded the volume.        -   7.3.2.11 Removed 200 μl for a cell count using an automated            cell counter.        -   7.3.2.12 While counting, placed the 50 ml conical tube with            TIL cells into a humidified, 5% CO₂, 37° C. incubator, with            the cap loosened to allow gas exchange.        -   7.3.2.13 Recorded the counts.        -   7.3.2.14 Removed the 50 ml conical tube containing the TIL            cells from the incubator and resuspend them cells at a            concentration of 1.3×10⁶ cells/ml in warm CM2 supplemented            with 30001 U/ml IL-2. Returned the 50 ml conical tube to the            incubator with a loosened cap.        -   7.3.2.15 If desired, kept the original 24-well plate to            reculture any residual TIL.        -   7.3.2.16 Repeated steps 7.3.2.4-7.3.2.15 for the second TIL            line.        -   7.3.2.17 Just prior to plating the TIL into the T25 flasks            for the experiment, TIL were diluted 1:10 for a final            concentration of 1.3×10⁵ cells/ml as per step 7.3.2.35            below.

Prepare MACS GMP CD3 Pure (OKT3) Working Solution

-   -   7.3.2.18 Took out stock solution of OKT3 (1 mg/ml) from 4° C.        refrigerator and placed in BSC.    -   7.3.2.19 A final concentration of 30 ng/ml OKT3 was used in the        media of the mini-REP.    -   7.3.2.20 600 ng of OKT3 were needed for 20 ml in each T25 flask        of the experiment; this was the equivalent of 60 μl of a 10        μg/ml solution for each 20 ml, or 360 μl for all 6 flasks tested        for each feeder lot.    -   7.3.2.21 For each feeder lot tested, made 400 μl of a 1:100        dilution of 1 mg/ml OKT3 for a working concentration of 10 μg/ml        (e.g., for testing 4 feeder lots at one time, make 1600 μl of a        1:100 dilution of 1 mg/ml OKT3: 16 μl of 1 mg/ml OKT3+1.584 ml        of CM2 medium with 30001 U/ml IL-2.)

Prepare T25 Flasks

-   -   7.3.2.22 Labeled each flask with the name of the TIL line        tested, flask replicate number, feeder lot number, date, and        initials of analyst.    -   7.3.2.23 Filled flask with the CM2 medium prior to preparing the        feeder cells.    -   7.3.2.24 Placed flasks into 37° C. humidified 5% CO₂ incubator        to keep media warm while waiting to add the remaining        components.    -   7.3.2.25 Once feeder cells were prepared, the components will be        added to the CM2 in each flask.

Prepare MACS GMP CD3 Pure (OKT3) Working Solution.

TABLE 21 Solutions Volume in Volume in control co-culture (feederComponent flasks only) flasks CM2 + 3000 IU/ml IL-2 18 ml 19 ml MNC: 1.3× 10⁷/ml in CM2 + 3000IU IL-2 1 ml 1 ml (final concentration 1.3 ×10⁷/flask) OKT3: 10 μg/ml in CM2 + 3000IU IL-2 60 μl 60 μl TIL: 1.3 ×10⁵/ml in CM2 with 3000IU of IL-2 1 ml 0 (final concentration 1.3 ×10⁵/flask)

Prepare Feeder Cells

-   -   7.3.2.26 A minimum of 78×10⁶ feeder cells were needed per lot        tested for this protocol. Each 1 ml vial frozen by SDBB had        100×10⁶ viable cells upon freezing. Assuming a 50% recovery upon        thaw from LN2 storage, it was recommended to thaw at least two 1        ml vials of feeder cells per lot giving an estimated 100×10⁶        viable cells for each REP. Alternately, if supplied in 1.8 ml        vials, only one vial provided enough feeder cells.    -   7.3.2.27 Before thawing feeder cells, pre-warmed approximately        50 ml of CM2 without IL-2 for each feeder lot to be tested.    -   7.3.2.28 Removed the designated feeder lot vials from LN2        storage, placed in zipper storage bag, and place on ice.        Transferred vials to tissue culture room.    -   7.3.2.29 Thawed vials inside closed zipper storage bag by        immersing in a 37° C. water bath.    -   7.3.2.30 Removed vials from zipper bag, spray or wipe with 70%        EtOH and transferred vials to BSC.    -   7.3.2.31 Using a transfer pipet immediately transferred the        contents of feeder vials into 30 ml of warm CM2 in a 50 ml        conical tube. Washed vial with a small volume of CM2 to remove        any residual cells in the vial.    -   7.3.2.32 Centrifuged at 400×CF for 5 minutes.    -   7.3.2.33 Aspirated the supernatant and resuspended in 4 ml warm        CM2 plus 3000 IU/ml IL-2.    -   7.3.2.34 Removed 200 μl for cell counting using the Automated        Cell Counter. Recorded the counts.    -   7.3.2.34 Resuspended cells at 1.3×10⁷ cells/ml in warm CM2 plus        3000 IU/ml IL-2.    -   7.3.2.34 Diluted TIL cells from 1.3×10⁶ cells/ml to 1.3×10⁵        cells/ml. Worked with each TIL line independently to prevent        cross-contamination.

Setup Co-Culture

-   -   7.3.2.36 Diluted TIL cells from 1.3×10⁶ cells/ml to 1.3×10⁵        cells/ml. Worked with each TIL line independently to prevent        cross-contamination.        -   7.3.2.36.1 Added 4.5 ml of CM2 medium to a 15 ml conical            tube.        -   7.3.2.36.2 Removed TIL cells from incubator and resuspended            well using a 10 ml serological pipet.        -   7.3.2.36.3 Removed 0.5 ml of cells from the 1.3×10⁶ cells/ml            TIL suspension and added to the 4.5 ml of medium in the 15            ml conical tube. Returned TIL stock vial to incubator.        -   7.3.2.36.4 Mixed well.        -   7.3.2.36.5 Repeated steps 7.3.2.36.1-7.3.2.36.4 for the            second TIL line.        -   7.3.2.36.6 If testing more than one feeder lot at one time,            diluted the TIL to the lower concentration for each feeder            lot just prior to plating the TIL.        -   7.3.2.37 Transferred flasks with pre-warmed media for a            single feeder lot from the incubator to the BSC.        -   7.3.2.38 Mixed feeder cells by pipetting up and down several            times with a 1 ml pipet tip and transferred 1 ml (1.3×10⁷            cells) to each flask for that feeder lot.        -   7.3.2.39 Added 60 μl of OKT3 working stock (10 μg/ml) to            each flask.        -   7.3.2.40 Returned the two control flasks to the incubator.        -   7.3.2.41 Transferred 1 ml (1.3×10⁵) of each TIL lot to the            correspondingly labeled T25 flask.        -   7.3.2.42 Returned flasks to the incubator and incubate            upright. Did not disturb until Day 5.        -   7.3.2.43 Repeated 7.3.2.36-7.3.2.42 for all feeder lots            tested.

Day 5, Media Change

-   -   7.3.3.1 Prepared CM2 with 3000 IU/ml IL-2. 10 ml is needed for        each flask    -   7.3.3.2 To prevent cross-contamination, handled the flasks for a        single feeder lot at a time. Removed flasks from the incubator        and transfer to the BSC, care was taken not to disturb the cell        layer on the bottom of the flask.    -   7.3.3.3 Repeated for all flasks including control flask.    -   7.3.3.4 With a 10 ml pipette, transferred 10 ml warm CM2 with        3000 IU/ml IL-2 to each flask.    -   7.3.3.5 Returned flasks to the incubator and incubate upright        until Day 7. Repeated 7.3.3.1-7.3.3.6 for all feeder lots        tested.

Day 7, Harvest

-   -   7.3.4.1 To prevent cross-contamination, handled the flasks for a        single feeder lot at a time.    -   7.3.4.2 Removed flasks from the incubator and transfer to the        BSC, care as taken not to disturb the cell layer on the bottom        of the flask.    -   7.3.4.3 Without disturbing the cells growing on the bottom of        the flasks, removed 10 ml of medium from each test flask and 15        ml of medium from each of the control flasks.    -   7.3.4.4 Using a 10 ml serological pipet, resuspended the cells        in the remaining medium and mix well to break up any clumps of        cells.    -   7.3.4.5 Recorded the volumes for each flask.    -   7.3.4.6 After thoroughly mixing cell suspension by pipetting,        removed 200 μl for cell counting.    -   7.3.4.7 Counted the TIL using the appropriate standard operating        procedure in conjunction with the automatic cell counter        equipment.    -   7.3.4.8 Recorded counts in Day 7.    -   7.3.4.9 Repeated 7.3.4.1-7.3.4.8 for all feeder lots tested.    -   7.3.4.10 Feeder control flasks were evaluated for replication        incompetence and flasks containing TIL were evaluated for fold        expansion from Day 0 according to the criteria listed in Table        21 (below).

Day 7, Continuation of Feeder Control Flasks to Day 14

-   -   7.3.5.1 After completing the Day 7 counts of the feeder control        flasks, added 15 ml of fresh CM2 medium containing 3000 IU/ml        IL-2 to each of the control flasks.    -   7.3.5.2 Returned the control flasks to the incubator and        incubated in an upright position until Day 14.

Day 14, Extended Non-Proliferation of Feeder Control Flasks

-   -   7.3.6.1 To prevent cross-contamination, handled the flasks for a        single feeder lot at a time.    -   7.3.6.2 Removed flasks from the incubator and transfer to the        BSC, care was taken not to disturb the cell layer on the bottom        of the flask.    -   7.3.6.3 Without disturbing the cells growing on the bottom of        the flasks, removed approximately 17 ml of medium from each        control flasks.    -   7.3.6.4 Using a 5 ml serological pipet, resuspended the cells in        the remaining medium and mixed well to break up any clumps of        cells.    -   7.3.6.5 Recorded the volumes for each flask.    -   7.3.6.6 After thoroughly mixing cell suspension by pipetting,        removed 200 μl for cell counting.    -   7.3.6.7 Counted the TIL using the appropriate standard operating        procedure in conjunction with the automatic cell counter        equipment.    -   7.3.6.8 Recorded counts.    -   7.3.6.9 Repeated 7.3.4.1-7.3.4.8 for all feeder lots tested.        Results and Acceptance Criteria

Results

-   -   10.1.1 The dose of gamma irradiation was sufficient to render        the feeder cells replication incompetent. All lots were expected        to meet the evaluation criteria and also demonstrated a        reduction in the total viable number of feeder cells remaining        on Day 7 of the REP culture compared to Day 0.    -   10.1.2 All feeder lots were expected to meet the evaluation        criteria of 100-fold expansion of TIL growth by Day 7 of the REP        culture.    -   10.1.3 Day 14 counts of Feeder Control flasks were expected to        continue the non-proliferative trend seen on Day 7.

Acceptance Criteria

-   -   10.2.1 The following acceptance criteria were met for each        replicate TIL line tested for each lot of feeder cells    -   10.2.2 Acceptance was two-fold, as follows (outlined in the        Table below).

TABLE 22 Acceptance Criteria Test Acceptance criteria Irradiation of Nogrowth observed at 7 and 14 days MNC/Replication Incompetence TILexpansion At least a 100-fold expansion of each TIL (minimum of 1.3 ×10⁷ viable cells)

-   -   -   10.2.2.1 Evaluated whether the dose of radiation was            sufficient to render the MNC feeder cells replication            incompetent when cultured in the presence of 30 ng/ml OKT3            antibody and 3000 IU/ml IL-2.            -   10.2.2.1.1 Replication incompetence was evaluated by                total viable cell count (TVC) as determined by automated                cell counting on Day 7 and Day 14 of the REP.            -   10.2.2.1.2 Acceptance criteria was “No Growth,” meaning                the total viable cell number has not increased on Day 7                and Day 14 from the initial viable cell number put into                culture on Day 0 of the REP.        -   10.2.2.2 Evaluated the ability of the feeder cells to            support TIL expansion.            -   10.2.2.2.1 TIL growth was measured in terms of fold                expansion of viable cells from the onset of culture on                Day 0 of the REP to Day 7 of the REP.            -   10.2.2.2.1 On Day 7, TIL cultures achieved a minimum of                100-fold expansion, (i.e., greater than 100 times the                number of total viable TIL cells put into culture on REP                Day 0), as evaluated by automated cell counting.        -   10.2.2.3 Should a lot fail to meet the two criteria above,            the lot was retested according to the contingency plan            outlined in Section 10.3 below.        -   10.2.2.4 Following retesting of a failed lot, any MNC feeder            lot that did not meet the two acceptance criteria in both            the original evaluation and the contingency testing was            excluded.        -   10.2.2.5 Any MNC feeder lots that meet acceptance criteria            but were judged to have poor performance in regard to the            ability to expand TIL relative to other previous feeder lots            tested in parallel with the same pre-REP TIL lines were            excluded.

Contingency Testing of MNC Feeder Lots that do not Meet AcceptanceCriteria

-   -   10.3.1 In the event that an MNC feeder lot did not meet the        either of the acceptance criteria outlined in Section 10.2        above, the following steps will be taken to retest the lot to        rule out simple experimenter error as its cause.    -   10.3.2 If there are two or more remaining satellite testing        vials of the lot, then the lot was retested. If there were one        or no remaining satellite testing vials of the lot, then the lot        was failed according to the acceptance criteria listed in        Section 10.2 above.    -   10.3.3 Two trained personnel, include the original person who        evaluated the lot in question, both tested the lot at the same        time.    -   10.3.4 Repeating Section 7.2-7.3 was done to re-evaluate the lot        in question.    -   10.3.5 Each person tested the lot in question as well as a        control lot (as defined in Section 7.2.4 above).    -   10.3.6 In order to be qualified, the lot in question and the        control lot had to achieve the acceptance criteria of Section        10.2 for both of the personnel doing the contingency testing.    -   10.3.7 Upon meeting these criteria, the lot was then released        for CMO use as outlined in Section 10.2 above.

Example 14: Qualifying Individual Lots of Gamma-Irradiated PeripheralBlood Mononuclear Cells

This Example describes a novel abbreviated procedure for qualifyingindividual lots of gamma-irradiated peripheral blood mononuclear cells(PBMC) for use as allogeneic feeder cells in the exemplary methodsdescribed herein. This example provides a protocol for the evaluation ofirradiated PBMC cell lots for use in the production of clinical lots ofTIL. Each irradiated PBMC lot was prepared from an individual donor.Over the course of more than 100 qualification protocols, it was beenshown that, in all cases, irradiated PBMC lots from SDBB (San DiegoBlood Bank) expand TIL >100-fold on Day 7 of a REP. This modifiedqualification protocol was intended to apply to irradiated donor PBMClots from SDBB which were then further tested to verify that thereceived dose of gamma radiation was sufficient to render themreplication incompetent. Once demonstrated that they maintainedreplication incompetence over the course of 14 days, donor PBMC lotswere considered “qualified” for usage to produce clinical lots of TIL.

Key Terms and Definitions

-   μgμMicrogram-   μl—Microliter-   AIM-V—commercially available cell culture medium Biological Safety    Cabinet-   BSC—Cluster of Differentiation-   CD—Complete Medium for TIL #2-   CM2—CM2 supplemented with 3000 IU/ml IL-2-   CM2IL2—Contract Manufacturing Organization-   CO₂—Carbon Dioxide-   EtOH—Ethanol-   GMP—Good Manufacturing Practices-   Gy—Gray-   IL—Interleukin-   IU—International Units-   LN2—Liquid Nitrogen-   MI—Milliliter-   NA—Not Applicable-   OKT3—anti-CD3 monoclonal antibody designation-   P20—2-20 μl pipettor-   P200—20-200 μl pipettor-   PBMC—peripheral blood mononuclear cells-   P1000—100-1000 μl pipettor-   PPE—Personal Protective Equipment-   REP—Rapid Expansion Protocol-   SDBB—San Diego Blood Bank-   TIL—Tumor Infiltrating Lymphocytes-   T25—25 cm2 tissue culture flask-   ×g—“times gravity”—measure of relative centrifugal force

Specimens Include Irradiated Donor PBMC (SDBB).

Procedure

Background

-   -   7.1.1 Gamma-irradiated, growth-arrested PBMC were required for        current standard REP of TIL. Membrane receptors on the PBMCs        bind to anti-CD3 (clone OKT3) antibody and crosslink to TIL in        culture, stimulating the TIL to expand. PBMC lots were prepared        from the leukapheresis of whole blood taken from individual        donors. The leukapheresis product was subjected to        centrifugation over Ficoll-Hypaque, washed, irradiated, and        cryopreserved under GMP conditions.        -   It is important that patients who received TIL therapy not            be infused with viable PBMCs as this could result in            Graft-Versus-Host Disease (GVHD). Donor PBMCs are therefore            growth-arrested by dosing the cells with gamma-irradiation,            resulting in double strand DNA breaks and the loss of cell            viability of the PBMCs upon reculture.            Evaluation Criteria    -   7.2.1 Evaluation criterion for irradiated PBMC lots was their        replication incompetency.        Experimental Set-Up    -   7.3.1 Feeder lots were tested in mini-REP format as if they were        to be co-cultured with TIL, using upright T25 tissue culture        flasks.        -   7.3.1.1 Control lot: One lot of irradiated PBMCs, which had            historically been shown to meet the criterion of 7.2.1, was            run alongside the experimental lots as a control.    -   7.3.2 For each lot of irradiated donor PBMC tested, duplicate        flasks was run.        Experimental Protocol

All tissue culture work in this protocol was done using steriletechnique in a BSC.

Day 0

-   -   7.4.1 Prepared ˜90 ml of CM2 medium for each lot of donor PBMC        to be tested. Kept CM2 warm in 37° C. water bath.    -   7.4.2 Thawed an aliquot of 6×10⁶ IU/ml IL-2.    -   7.4.3 Returned the CM2 medium to the BSC, wiping with 70% EtOH        prior to placing in hood. For each lot of PBMC tested, removed        about 60 ml of CM2 to a separate sterile bottle. Added IL-2 from        the thawed 6×10⁶ IU/ml stock solution to this medium for a final        concentration of 3000 IU/ml. Labeled this bottle as “CM2/IL2”        (or similar) to distinguish it from the unsupplemented CM2.    -   7.4.4 Labeled two T25 flasks for each lot of PBMC to be tested.        Minimal label included:        -   7.4.4.1 Lot number        -   7.4.4.2 Flask number (1 or 2)        -   7.4.4.3 Date of initiation of culture (Day 0)            Prepare OKT3    -   7.4.5 Took out the stock solution of anti-CD3 (OKT3) from the        4° C. refrigerator and placed in the BSC.    -   7.4.6 A final concentration of 30 ng/ml OKT3 was used in the        media of the mini-REP.    -   7.4.7 Prepared a 10 μg/ml working solution of anti-CD3 (OKT3)        from the 1 mg/ml stock solution. Placed in refrigerator until        needed.        -   7.4.7.1 For each PBMC lot tested, prepare 150 μl of a 1:100            dilution of the anti-CD3 (OKT3) stock            -   E.g., for testing 4 PBMC lots at one time, prepare 600                μl of 10 μg/ml anti-CD3 (OKT3) by adding 6 W of the 1                mg/ml stock solution to 594 μl of CM2 supplemented with                3000 IU/ml IL-2.                Prepare Flasks    -   7.4.8 Added 19 ml per flask of CM2/IL-2 to the labeled T25        flasks and placed flasks into 37° C., humidified, 5% CO₂        incubator while preparing cells.        Prepare Irradiate PBMC    -   7.4.9 Worked with each donor PBMC lot individually to avoid the        potential cross-contamination of the lots.    -   7.4.10 Retrieved vials of PBMC lots to be tested from LN2        storage. These were placed at −80° C. or kept on dry ice prior        to thawing.    -   7.4.11 Placed 30 ml of CM2 (without IL-2 supplement) into 50 ml        conical tubes for each lot to be thawed. Labeled each tube with        the different lot numbers of the PBMC to be thawed. Capped tubes        tightly and place in 37° C. water bath prior to use. As needed,        returned 50 ml conical tubes to the BSC, wiping with 70% EtOH        prior to placing in the hood.    -   7.4.12 Removed a vial PBMC from cold storage and place in a        floating tube rack in a 37° C. water bath to thaw. Allowed thaw        to proceed until a small amount of ice remains in the vial.    -   7.4.13 Sprayed or wiped thawed vial with 70% EtOH and transfer        to BSC.    -   7.4.14 Using a sterile transfer pipet, immediately transferred        the contents of the vial into the 30 ml of CM2 in the 50 ml        conical tube. Removed about 1 ml of medium from the tube to        rinse the vial; returned rinse to the 50 ml conical tube. Capped        tightly and swirl gently to wash cells.    -   7.4.15 Centrifuged at 400×g for 5 min at room temperature.    -   7.4.16 Aspirated the supernatant and resuspend the cell pellet        in 1 ml of warm CM2/IL-2 using a 1000 μl pipet tip. Alternately,        prior to adding medium, resuspended cell pellet by dragging        capped tube along an empty tube rack. After resuspending the        cell pellet, brought volume to 4 ml using CM2/IL-2 medium.        Recorded volume.    -   7.4.17 Removed a small aliquot (e.g., 100 μl) for cell counting        using an automated cell counter.        -   7.4.17.1 Performed counts in duplicate according to the            particular automated cell counter SOP. It most likely was            necessary to perform a dilution of the PBMC prior to            performing the cell counts. A recommended starting dilution            was 1:10, but this varied depending on the type of cell            counter used.        -   7.4.17.2 Recorded the counts.    -   7.4.18 Adjusted concentration of PBMC to 1.3×10⁷ cells/ml as per        the worksheet in step 7.4.15.2 using CM2/IL-2 medium. Mixed well        by gentle swirling or by gently aspirating up-and-down using a        serological pipet.        Set Up Culture Flasks    -   7.4.19 Returned two labeled T25 flasks to the BSC from the        tissue culture incubator.    -   7.4.20 Returned the 10 μg/ml vial of anti-CD3/OKT3 to the BSC.    -   7.4.21 Added 1 ml of the 1.3×10⁷ PBMC cell suspension to each        flask.    -   7.4.22 Added 60 μl of the 10 μg/ml anti-CD3/OKT3 to each flask.    -   7.4.23 Returned capped flasks to the tissue culture incubators        for 14 days of growth without disturbance.    -   7.4.24 Placed anti-CD3/OKT3 vial back into the refrigerator        until needed for the next lot.    -   7.4.25 Repeated steps 7.4.9-7.4.24 for each lot of PBMC to be        evaluated.        Day 14, Measurement of Non-Proliferation of PBMC    -   7.4.26 Working with each lot independently, carefully returned        the duplicate T25 flasks to the BSC.    -   7.4.27 For each flask, using a fresh 10 ml serological pipet,        removed ˜17 ml from each of the flasks, then carefully pulled up        the remaining media to measure the volume remaining in the        flasks. Recorded volume.    -   7.4.28 Mixed sample well by pipetting up and down using the same        serological pipet.    -   7.4.29 Removed a 200 μl sample from each flask for counting.    -   7.4.30 Counted cells using an automated cell counter.    -   7.4.31 Repeated steps 7.4.26-7.4.31 for each lot of PBMC being        evaluated.        Results and Acceptance Criterion        Results    -   10.1.1 The dose of gamma irradiation was expected to be        sufficient to render the feeder cells replication incompetent.        All lots were expected to meet the evaluation criterion,        demonstrating a reduction in the total viable number of feeder        cells remaining on Day 14 of the REP culture compared to Day 0.        Acceptance Criterion    -   10.2.1 The following acceptance criterion were met for each        irradiated donor PBMC lot tested:    -   10.2.2 “No growth”—meant that the total number of viable cells        on Day 14 was less than the initial viable cell number put into        culture on Day 0 of the REP.    -   10.2.3 Should a lot fail to meet the criterion above, the lot        was retested per the Contingency Testing Procedure outlined in        the section 10.4.    -   10.2.4 Following retesting of a failed lot, any MNC feeder lot        that did not meet the acceptance criterion in both the original        evaluation and the contingency testing was excluded.        Contingency Testing of PBMC Lots which do not Meet Acceptance        Criterion.    -   10.4.1 In the event than an irradiated donor PBMC lot did not        meet the acceptance criterion above, the following steps were        taken to retest the lot to rule out simple experimenter error as        the cause of its failure.    -   10.4.2 If there were two or more remaining satellite vials of        the lot, then the lot was retested. If there are one or no        remaining satellite vials of the lot, then the lot was failed        according to the acceptance criterion of section 10.2 above.    -   10.4.3 Whenever possible, two trained personnel (preferably        including the original person who evaluated the lot in question)        did the testing of the two separate vials independently. This        was the preferred method of contingency testing. Aside from the        separate vials of PBMC, the same reagents could be used by both        personnel.        -   10.4.3.1. If two personnel were not available, one person            did the testing of the two PBMC vials for the failed lot,            working with each vial independently.        -   10.4.4 Repeating of section 7.4 “Experimental Protocol” was            done to re-evaluated the lot in question.        -   10.4.5 In addition to the lot in question, a control lot was            tested by each person carrying out the contingency testing.            -   10.4.5.1 If two personnel perform contingency testing,                both personnel tested the control lot independently.            -   10.4.5.2 If only one person is available to perform                contingency testing, it was not necessary for the                control lot to be run in duplicate.            -   10.4.5.3 To be qualified, a PBMC lot going through                contingency testing had both the control lot and both                replicates of the lot in question achieve the acceptance                criterion of Section 10.2 to pass.            -   10.4.5.4 Upon meeting this criterion, the lot was then                released for CMO usage as outlined in section 10.2.

Example 15: Cellometer IC2 Image Cytometer Automatic Cell Counter

This Example describes the procedure for operation of the Cellometer K2Image Cytometer automatic cell counter.

1. Definitions

-   -   μl Microliter    -   AOPI Acridine Orange Propidium Iodine    -   BSC Biological Safety Cabinet    -   DPBS Dulbecco's Phosphate Buffered Saline    -   ml Milliliter    -   MNC Mononuclear Blood Cells    -   NA Not Applicable    -   PBMC Peripheral Blood Mononuclear Cells    -   PPE Personal Protective Equipment    -   Pre-REP Initial TIL culture before Rapid Expansion Protocol of        culture    -   REP Rapid Expansion Protocol    -   TIL Tumor Infiltrating Lymphocytes

7. Procedure

-   -   7.1 Cell suspension preparation        -   7.1.1 Trypan Blue Preparation        -   The final Trypan blue concentration was 0.1%. The            manufacturer recommended preparing a stock solution of 0.2%.            -   7.1.1.1 When using Trypan blue on the Cellometer K2,                diluted the stock (0.4%) with PBS to 0.2%.            -   7.1.1.2 Filtered the Trypan blue with a 0.2-0.4 micron                filter and aliquot in small volumes into labeled, capped                tubes.            -   7.1.1.3 Mixed the cell suspension at 1:1 with 0.2%                trypan blue.        -   7.1.2 AOPI Preparation            -   7.1.2.1 When using AOPI on the Cellometer K2, obtained                the AOPI solution.            -   7.1.2.2 Stained cell sample at 1:1 with AOPI solution.        -   NOTE: When counting high concentration cultures, diluted the            cell samples in cell culture medium prior to the final 1:1            dilution with Trypan Blue or AOPI.        -   Used manufacturer's suggested range of counting to determine            the best dilution to use.    -   7.2 Cellometer K2 Set-Up        -   7.2.1 Turned on the Cellometer K2 equipment.        -   7.2.2 Selected the Cellometer Image Cytometer icon on the            associated computer monitor.        -   7.2.3 On the main screen of the software, selected one of            the Assays listed in the dropdown box.            -   7.2.3.1 When selecting the appropriate Assay, the Cell                Type and Image Mode self-populated.            -   7.2.3.2 Under “Sample” section, clicked on Set                User/Sample ID to open another screen to input                operator's information for specimen.                -   7.2.3.2.1 Entered “User ID”. This will consist of                    the user's three letter initials.                -   7.2.3.2.2 Entered “Sample ID”. The sample ID is                    derived from incoming specimen information.            -   7.2.3.3 Set up dilution parameters.                -   7.2.3.3.1 If no other dilution was made besides the                    1:1 mixture, the dilution factor was 2.                -   7.2.3.3.2 If a dilution was made prior to the final                    1:1 mixture, the dilution factor was 2 times of the                    prior dilution.                -   7.2.3.3.3 Updated dilution factor according to the                    mixture used in the dilution section of the screen.                    Clicked on the pencil icon to bring up the dialog                    screens.                -   7.2.3.3.4 Verified that F1 Image and F2 Image                    sections are identical to each other.                -   7.2.3.3.5 Clicked on the “Save” button after set up                    has been completed.    -   7.3 Cell Counting        -   7.3.1 Removed the plastic backing from both sides of a            Cellometer counting chamber slide (SD100) and placed it on            top of a clean, lint-free wipe.        -   7.3.2 After preparing the cell suspension, removed a small            aliquot of the sample and transferred it into a well of a            multiwell cell culture plate or tube.        -   7.3.3 If diluting the sample, performed the dilution using            cell culture medium.        -   7.3.4 Added 20 μl of cell suspension into a well of the            multiwell cell culture plate or tube.        -   7.3.5 Added 20 111 of 0.2% trypan blue or the AOPI solution            to the 20111 of cell suspension and mix sample thoroughly.        -   7.3.6 Measured 20 IA of the 1:1 solution and transferred it            into one side of the counting chamber.    -   NOTE: Avoided touching the clear area of the slide.        -   7.3.7 If necessary, repeated the sample on the other side of            the slide. 7.3.8. Inserted the chamber into the slot on the            front of the Cellometer.        -   7.3.8 For the AOPI cell counting, clicked on “Preview Fl” on            the main screen to preview the green fluorescent image (live            cell) image. For Trypan blue counting, clicked on “Preview            Brightfield”.        -   7.3.9 Using the focusing wheel, brought image into optimal            focus. Cells had a bright center and a clearly-defined edge.        -   7.3.10 Clicked “Count” to begin the counting process.        -   7.3.11 Results were displayed in a counting results pop-up            box on the computer screen showing the results of the            counting process.

Example 16: Preparation of IL-2 Stock Solution (Cellgenix)

This Example describes the process of dissolving purified, lyophilizedrecombinant human interleukin-2 into stock samples suitable for use infurther tissue culture protocols, including all of those described inthe present application and Examples, including those that involve usingrhIL-2.

3. Definitions/Abbreviations

-   -   μL: microliter    -   BSC: Biological Safety Cabinet    -   BSL2: Biosafety Level 2    -   D-PBS: Dulbecco's Phosphate Buffered Saline    -   G: Gauge    -   GMP: Good Manufacturing Processing    -   HAc: Acetic Acid    -   HSA: Human Serum Albumin    -   mL: Milliliter    -   NA: Not applicable    -   PPE: Personal Protective Equipment    -   rhIL-2; IL-2: Recombinant human Interleukin-2    -   COA: Certificate of Analysis

6. Procedure

-   -   6.1 Prepare 0.2% Acetic Acid solution (HAc).        -   6.1.1 Transferred 29 mL sterile water to a 50 mL conical            tube.        -   6.1.2 Added 1 mL 1N acetic acid to the 50 mL conical tube.        -   6.1.3 Mixed well by inverting tube 2-3 times.        -   6.1.4 Sterilized the HAc solution by filtration using a            Steriflip filter.        -   6.1.5 Capped, dated, and labeled the solution “Sterile 0.2%            Acetic Acid Solution”        -   6.1.6 Solution expired after 2 months. Stored at room            temperature.    -   6.2 Prepare 1% HSA in PBS.        -   6.2.1 Added 4 mL of 25% HSA stock solution to 96 mL PBS in a            150 mL sterile filter unit.        -   6.2.2 Filtered solution.        -   6.2.3 Capped, dated, and labeled the solution “1% HSA in            PBS.”        -   6.2.4 Solution expired after 2 months. Store 4° C.    -   6.3 For each vial of rhIL-2 prepared, fill out forms.    -   6.4 Prepared rhIL-2 stock solution (6×10⁶ IU/mL final        concentration)        -   6.4.1 Each lot of rh1 L-2 was different and required            information found in the manufacturer's Certificate of            Analysis (COA), such as:            -   6.4.1.1 Mass of rhIL-2 per vial (mg)            -   6.4.1.2 Specific activity of rhIL-2 (IU/mg)            -   6.4.1.3 Recommended 0.2% HAc reconstitution volume (mL)        -   6.4.2 Calculated the volume of 1% HSA required for rhIL-2            lot by using the equation below:

${\left( \frac{\begin{matrix}{{Vial}\mspace{14mu}{{Mass}{\mspace{11mu}\;}({mg})} \times} \\{{Biological}\mspace{14mu}{Activity}\mspace{11mu}\left( \frac{IU}{mg} \right)}\end{matrix}}{6 \times 10^{6}\frac{IU}{mL}} \right) - {{HAc}\mspace{14mu}{vol}\mspace{14mu}({mL})}} = {1\%\mspace{14mu}{HSA}\mspace{14mu}{vol}\mspace{14mu}({mL})}$

-   -   -   -   6.4.2.1 For example, according to CellGenix's rhIL-2 lot                10200121 COA, the specific activity for the 1 mg vial is                25×10⁶ IU/mg. It recommends reconstituting the rhIL-2 in                2 mL 0.2% HAc.

${\left( \frac{1\mspace{14mu}{mg} \times 25 \times 10^{6}\frac{IU}{mg}}{6 \times 10^{6}\frac{IU}{mL}} \right) - {2\mspace{14mu}{mL}}} = {2.167\mspace{14mu}{mL}\mspace{14mu}{HSA}}$

-   -   -   6.4.3 Wiped rubber stopper of IL-2 vial with alcohol wipe.        -   6.4.4 Using a 16 G needle attached to a 3 mL syringe,            injected recommended volume of 0.2% HAc into vial. Took care            to not dislodge the stopper as the needle is withdrawn.        -   6.4.5 Inverted vial 3 times and swirled until all powder is            dissolved.        -   6.4.6 Carefully removed the stopper and set aside on an            alcohol wipe.        -   6.4.7 Added the calculated volume of 1% HSA to the vial.        -   6.4.8 Capped the vial with the rubber stopper.

    -   6.5 Storage of rhIL-2 solution        -   6.5.1 For short-term storage (<72 hrs), stored vial at 4° C.        -   6.5.2 For long-term storage (>72 hrs), aliquoted vial into            smaller volumes and stored in cryovials at −20° C. until            ready to use. Avoided freeze/thaw cycles. Expired 6 months            after date of preparation.        -   6.5.3 Rh-IL-2 labels included vendor and catalog number, lot            number, expiration date, operator initials, concentration            and volume of aliquot.

Example 17: Preparation of Media for Pre-Rep and Rep Processes

This Example describes the procedure for the preparation of tissueculture media for use in protocols involving the culture of tumorinfiltrating lymphocytes (TIL) derived from various tumor typesincluding, but not limited to, metastatic melanoma, head and necksquamous cell carcinoma (HNSCC), ovarian carcinoma, triple-negativebreast carcinoma, and lung adenocarcinoma. This media can be used forpreparation of any of the TILs described in the present application andExamples.

3. Definition

-   -   μg microgram    -   μm micrometer    -   μM micromolar    -   AIM-V® serum-free tissue culture medium (Thermo Fisher        Scientific)    -   BSC Biological Safety Cabinet    -   CM1 Complete Medium #1    -   CM2 Complete Medium #2    -   CM3 Complete Medium #3    -   CM4 Complete Medium #4    -   IU or U International units    -   ml milliliter    -   mM millimolar    -   NA not applicable    -   PPE personal protective equipment    -   Pre-REP pre-Rapid Expansion Process    -   REP Rapid Expansion Process    -   rhIL-2, IL-2 recombinant human Interleukin-2    -   RPMI1640 Roswell Park Memorial Institute medium, formulation        1640    -   SOP Standard Operating Procedure    -   TIL tumor infiltrating lymphocytes

7. Procedure

-   -   7.1 All procedures are done using sterile technique in a BSC        (Class II, Type A2).        -   7.1.1 Sprayed surface of hood with 70% ethanol prior to its            use.        -   7.1.2 Sprayed all items and reagents with 70% ethanol prior            to placing them into tissue culture hood.    -   7.2 Aliquotting of 200 mM L-glutamine        -   7.2.1 L-glutamine was supplied in larger volumes than needed            for the preparation of serum (e.g., 100 ml or 500 ml            volumes).        -   7.2.2 Thawed bottle of L-glutamine in 37° C. water bath.        -   7.2.3 Mixed L-glutamine well after thawing, as it            precipitates after thaw. Ensured that all precipitates have            returned to solution prior to aliquotting.        -   7.2.4 Placed 5-10 ml aliquots of L-glutamine into sterile 15            ml conical tubes.        -   7.2.5 Labeled tubes with concentration, vendor, lot number,            date aliquotted, and expiration date.        -   7.2.6 Tubes were then stored at −20° C. and pulled as needed            for media preparation.    -   7.3 Preparation of CM1        -   7.3.1 Removed the following reagents from cold storage and            warmed them in a 37° C. water bath:            -   7.3.1.1 RPMI1640            -   7.3.1.2 Human AB serum            -   7.3.1.3 200 mM L-glutamine        -   7.3.2 Removed the BME from 4° C. storage and place in tissue            culture hood.        -   7.3.3 Placed the gentamycin stock solution from room            temperature storage into tissue culture hood.        -   7.3.4 Prepared CM1 medium according to Table 23 below by            adding each of the ingredients into the top section of a 0.2            um filter unit appropriate to the volume to be filtered.

TABLE 23 Preparation of CM1 Final Final Volume Final Ingredientconcentration 500 ml Volume IL RPMI1640 NA 450 ml  900 ml  Human ABserum, 50 ml 100 ml  heat-inactivated 10% 200 mM L-glutamine  2 mM   5ml 10 ml  55 mM BME 55 μM 0.5 ml 1 ml 50 mg/ml gentamicin 50 μg/ml 0.5ml 1 ml sulfate

-   -   -   7.3.5 Labeled the CM1 media bottle with its name, the            initials of the preparer, the date it was filtered/prepared,            the two week expiration date and store at 4° C. until needed            for tissue culture. Media can be aliquotted into smaller            volume bottles as required.        -   7.3.6 Any remaining RPMI1640, Human AB serum, or L-glutamine            was stored at 4° C. until next preparation of media.        -   7.3.7 Stock bottle of BME was returned to 4° C. storage.        -   7.3.8 Stock bottle of gentamicin was returned to its proper            RT storage location.        -   7.3.9 Because of the limited buffering capacity of the            medium, CM1 was discarded no more than two weeks after            preparation, or as the phenol red pH indicator showed an            extreme shift in pH (bright red to pink coloration).        -   7.3.10 On the day of use, prewarmed required amount of CM1            in 37° C. water bath and add 6000 IU/ml IL-2.        -   7.3.11 Additional supplementation—as needed            -   7.3.11.1 CM1 supplemented with GlutaMAX®                -   7.3.11.1.1 CM1 could be prepared by substituting 2                    mM GlutaMAX™ for 2 mM glutamine (final                    concentration, see Table 2.) If this was done,                    labeled the media bottle as in Step 7.3.5 above                    adding “2 mM GlutaMAX” to prevent confusion with the                    standard formulation of CM1.            -   7.3.11.2 CM1 supplemented with extra                antibiotic/antimycotic                -   7.3.11.2.1 Some CM1 formulations required additional                    antibiotic or antimycotic to prevent contamination                    of pre-REP TIL grown from certain tumor types.                -   7.3.11.2.2 Added antibiotic/antimycotic to the final                    concentrations shown in Table 24 below.                -   7.3.11.2.3 If this was done, label the media bottle                    as in Step 7.3.1 above adding the name/s of the                    additional antibiotic/antimycotic to prevent                    confusion with the standard formulation of CM1.

TABLE 24 Additional supplementation of CM1, as needed. Stock FinalSupplement concentration Dilution concentration GlutaMAXTm 200 mM 1:100  2 mM Penicillin/streptomycin 10,000 U/ml 1:100 100 U/ml penicillinpenicillin 100 μg/ml 10,000 μg/ml streptomycin streptomycin AmphotericinB 250 μg/ml 1:100 2.5 μg/ml

-   -   7.4 Preparation of CM2        -   7.4.1 Removed prepared CM1 from refrigerator or prepare            fresh CM1 as per Section 7.3 above.        -   7.4.2 Removed AIM-V® from refrigerator.        -   7.4.3 Prepared the amount of CM2 needed by mixing prepared            CM1 with an equal volume of AIM-V® in a sterile media            bottle.        -   7.4.4 Added 3000 IU/ml IL-2 to CM2 medium on the day of            usage.        -   7.4.5 Made sufficient amount of CM2 with 3000 IU/ml IL-2 on            the day of usage.        -   7.4.6 Labeled the CM2 media bottle with its name, the            initials of the preparer, the date it was filtered/prepared,            the two week expiration date and store at 4° C. until needed            for tissue culture. Media was aliquotted into smaller volume            bottles as required.        -   7.4.7 Returned any CM2 without IL-2 to the refrigerator            where it can be stored for up to two weeks, or until phenol            red pH indicator shows an extreme shift in pH (bright red to            pink coloration).    -   7.5 Preparation of CM3        -   7.5.1 Prepared CM3 on the day it was required for use.        -   7.5.2 CM3 was the same as AIM-V® medium, supplemented with            3000 IU/ml IL-2 on the day of use.        -   7.5.3 Prepared an amount of CM3 sufficient to experimental            needs by adding IL-2 stock solution directly to the bottle            or bag of AIM-V. Mixed well by gentle shaking. Label bottle            with “3000 IU/ml IL-2” immediately after adding to the            AIM-V. If there was excess CM3, stored it in bottles at            4° C. labeled with the media name, the initials of the            preparer, the date the media was prepared, and its            expiration date (7 days after preparation).        -   7.5.4 Discarded media supplemented with IL-2 after 7 days            storage at 4° C.    -   7.6 Preparation of CM4        -   7.6.1 CM4 was the same as CM3, with the additional            supplement of 2 mM GlutaMAX™ (final concentration).            -   7.6.1.1 For every 1 L of CM3, added 10 ml of 200 mM                GlutaMAX™.        -   7.6.2 Prepared an amount of CM4 sufficient to experimental            needs by adding IL-2 stock solution and GlutaMAX™ stock            solution directly to the bottle or bag of AIM-V. Mixed well            by gentle shaking.        -   7.6.3 Labeled bottle with “3000 IL/nil IL-2 and GlutaMAX”            immediately after adding to the AIM-V.        -   7.6.4 If there was excess CM4, stored it in bottles at 4° C.            labeled with the media name, “GlutaMAX”, the initials of the            preparer, the date the media was prepared, and its            expiration date (7 days after preparation).        -   7.6.5 Discarded media supplemented with IL-2 after 7 days            storage at 4° C.

Example 18: Surface Antigen Staining of Post Rep TIL 1. Purpose

The Example describes the procedure for cell surface staining ofpost-REP TILs by flow cytometry. This procedure can be applied to anyTILs described in the application and Examples.

Key Terms and Definitions

-   -   α: Alpha    -   β: Beta    -   μl: Microliter    -   APC: Allophycocyanin    -   Ax647: Alex Fluor 647    -   BD: Becton Dickinson Company    -   BSA: Bovine Serum Albumin    -   BSC: Biological Safety Cabinet    -   BV421: Brilliant Violet 421    -   CD: Cluster of Differentiation    -   CST: Cytometer Setup and Tracking    -   Cy: Cyanine    -   DPBS: Dulbecco's Phosphate Buffered Saline    -   FACS: Fluorescence Activated Cell Sorter    -   FBS: Fetal Bovine Serum    -   FITC: Fluorescein Isothiocyanate    -   FMO: Fluorescence Minus One    -   G: Gram    -   H7: Analog of Cy7    -   Ml: Milliliter    -   PE: Phycoerythrin    -   PerCP-Cy5.5: Peridinin-Chlorophyll proteins    -   PPE: Personal Protective Equipment    -   REP: Rapid Expansion Protocol    -   SIT: Sample Injection Tube    -   TCR: T Cell Receptor    -   w/v: Weight to Volume        Flow Cytometry Antibodies and Stains

TABLE 25 Live/Dead Aqua Stain ThermoFisher Catalog # L34966. CatalogTarget Format Clone Supplier Number TCRab PE/Cy7 IP26 BioLegend 306720(i.e., TCRα/β) CD57 PerCP-Cy5.5 HNK-1 BioLegend 359622 CD28 PE CD28.2BioLegend 302908 CD4 FITC OKT4 eBioscience 11-0048-42 CD27 APC-H7 M-T271BD Biosciences 560222 CD56 APC N901 Beckman IM2474U Coulter CD8a PBRPA-T8 BioLegend 301033 CD45R A PE-Cy7 HI100 BD Biosciences 560675 CD8aPerCP/Cy5.5 RPA-T8 BioLegend 301032 CCR7 PE 150503 BD Biosciences 560765CD3 APC/Cy7 HIT3a BioLegend 300318 CD38 APC HB-7 BioLegend 356606 HLA-DRPB L243 BioLegend 307633 CD69 PE-Cy7 FN50 BD Biosciences 557745 TIGIT PEMBSA43 eBioscience 12-9500-42 KLRG1 Ax647 SA231A2 BioLegend 367704 CD154BV421 TRAP1 BD Biosciences 563886 CD137 PE/Cy7 4B4-1 BioLegend 309818Lag3 PE 3DS223H eBioscience 12-2239-42 PD1 APC EH12.2H 7 BioLegend329908 Tim-3 BV421 F38-2E2 BioLegend 345008

7. Procedure

-   -   7.1 Reagent Preparation        -   7.1.1 FACS Wash Buffer            -   7.1.1.1 Added 2% (w/v) heat-inactivated FBS to DPBS (Add                10 ml FBS to 490 mLs of 1×dPBS).            -   7.1.1.2 Added 0.1% (w/v) NaN₃ (76.9 ul to 500 mL                bottle.)            -   7.1.1.3 Solution was stored at 40° C. Discard after 30                days.        -   7.1.2 Aqua dye            -   7.1.2.1 Added 50 μl of DMSO to the vial of reactive dye.            -   7.1.2.2 Mixed well and visually confirm that all of the                dye has dissolved.            -   7.1.2.3 Dye that was not used for the procedure was                aliquoted and frozen at 20° C. until the next use. Did                not freeze/thaw a second time.        -   7.1.3 Antibody Cocktail Preparation.            -   7.1.3.1 Cocktails were made up in polypropylene tubes                such as an Eppendorf tube            -   7.1.3.2 Cocktails were stored for up to 6 months.

TABLE 26 Differentiation Panel 1 (DF1): Catalog Target Format CloneSupplier Number Titre TCRab PE/Cy7 IP26 BioLegend 306720 3 (i.e.,TCRα/β) CD57* PerCP- HNK-1 BioLegend 359622 2 Cy5.5 CD28* PE CD28.2BioLegend 302908 2 CD4 FITC OKT4 eBioscience 11-0048-42 2 CD27* APC-H7M-T271 BD 560222 3 Biosciences CD56 APC N901 Beckman IM2474U 3 CoulterCD8a PB RPA-T8 BioLegend 301033 2 FACS 33 Buffer

TABLE 27 Differentiation Panel 2 (DF2): Catalog Target Format CloneSupplier Number Titre CD45RA* PE-Cy7 HI100 BD 560675 1 Biosciences CCD3PerCP/Cy5.5 SP34-2 BD 552852 2 Biosciences CCCR7* PE 150503 BD 560765 5Biosciences CCD8 FITC HIT8 BioLegend 300906 2 CCD4 APC/Cy7 OKT4BioLegend 317418 2 CCD38* APC HB-7 BioLegend 356606 1 HHLA-DR PB L243BioLegend 307633 2 FACS 35 Buffer

TABLE 28 T-cell Activation Panel 1(Tact1) Catalog Target Format CloneSupplier Number Titre CD137* PE/Cy7 4B4-1 BioLegend 309818 2 CD3PerCP/Cy5.5 SP34-2 BD 552852 2 Biosciences Lag3* PE 3DS223H eBioscience12- 5 2239-42 CD8 FITC HIT8 BioLegend 300906 2 CD4 APCCy7 OKT4 BioLegend317418 2 PD1* APC EH12.2H7 BioLegend 329908 2 Tim-3* BV421 F38-2E2BioLegend 345008 2 FACS 33 Buffer

TABLE 29 T-cell Activation Panel 2(Tact2) Catalog Target Format CloneSupplier Number Titre CD69* PE-Cy7 FN50 BD 557745 3 Biosciences CD3PerCP/Cy5.5 SP34-2 BD 552852 2 Biosciences TIGIT* PE MBSA43 eBioscience12- 3 9500-42 CD8 FITC HIT8 BioLegend 300906 2 CD4 APCCy7 OKT4 BioLegend317418 2 KLRG1* Ax647 SA231A2 BioLegend 367704 1 CD154* BV421 TRAP1 BD563886 3 Biosciences FACS 34 Buffer

-   -   7.2 Flow Cytometry Assay Requirements        -   7.2.1 Flow Cytometer Calibration            -   7.2.1.1 The flow cytometer was calibrated on the day of                the assay using CST beads following manufacturer's                instructions.            -   7.2.1.2 The operator ensured that the flow cytometer had                passed calibration, where performance and baseline                checks are valid.        -   7.2.2 Compensation/FMO Controls            -   7.2.2.1 Single color compensation samples were prepared                using the BD compensation beads and the ArC™ Amine                Reactive Compensation Bead Kit.            -   7.2.2.2 FMO control, cell containing samples were                stained with a cocktail of antibodies minus the                following single antibody conjugate, CD27, CD28, and                CD57.        -   7.2.3 MFI Standardization            -   7.2.3.1 Cytometer voltages was determined daily with a                bead control and target voltage values.    -   7.3 Sample Staining        -   7.3.1 Labeled FACS tube with the Sample ID-DF1, Sample            ID-DF2, Sample ID-T1, Sample ID-T2.        -   7.3.2 Labeled one set of FMO controls with CD27-APC-H7,            CD28-PE, CD57-PerCPCy5.5, CD45RA-PECy7, CCR7-PE, CD38-APC,            CD137-PE7, Lag3-PE, PD1 APC, Tim3-BV421, CD69-PE7, TIGIT-PE,            KLRG1-Ax647, and CD154-BV421.        -   7.3.3 Added 0.5 to 2 million cells to each tube.        -   7.3.4 QS to 3 mLs of 1×PBS to each tube.        -   7.3.5 Spun the tubes at 400×g, high acceleration and brake,            for 5 minutes.        -   7.3.6 While the samples were centrifuging, prepared the dead            cell labeling Aqua dye.        -   7.3.7 Removed an Aqua aliquot from the freezer and dilute            1/200 in PBS. Keep dark. Add 2 uL dye to 198 uL DPBS.        -   7.3.8 Decanted or aspirated the supernatant from step 7.3.5.        -   7.3.9 Added 25 uL of Aqua solution from above to samples and            FMO controls.        -   7.3.10 Incubated for 15 minutes at Room Temperature (RT) in            the dark.        -   7.3.11 Note: If cells were initially stored in a protein            free media, then a blocking step should be added, such as 5            uL TruStain for 10 minutes at room temperature.        -   7.3.12 Added 50 uL of antibody cocktails to appropriate            tubes.        -   7.3.13 Shook tube rack to mix.        -   7.3.14 Incubated for 15 minutes at RT in the dark.        -   7.3.15 Recording starting and ending times. Added 3 mL of            FACS Wash buffer.        -   7.3.16 Spun tubes at 400×g, high acceleration and brake, for            5 minutes.        -   7.3.17 When centrifuge spin was complete, decanted or            aspirated the supernatant.        -   7.3.18 Resuspended cells by sliding the tubes along an empty            rack.        -   7.3.19 Added 100 uL of 1% ParaFormaldehyde to each tube.        -   7.3.20 Stored at 40 C in dark until ready to collect on Flow            Cytometer.

Note: Samples could be stored for up to 72 hours.

-   -   7.4 L/D Aqua compensation control.        -   7.4.1 Labeled FACS tubes as L/D Aqua compensation control.        -   7.4.2 Added one drop of Arc beads to the tube.        -   7.4.3 Added 3 μl of LID Aqua directly to the beads.        -   7.4.4 Incubated the tubes at room temperature in the dark            for 10 to 30 min.        -   7.4.5 Recorded starting and ending incubation time on the            worksheet        -   7.4.6 After incubation, added 3 ml of FACS Wash to each            tube.        -   7.4.7 Spun tubes at 400×g, high acceleration and brake, for            5 minutes.        -   7.4.8 Decanted or aspirated the supernatant.        -   7.4.9 Resuspended the tubes with 500 μl of 1% PFA solution.            Added 1 drop of negative bead. Placed at 40° C. in dark            until collection.    -   7.5 Compensation control staining.        -   7.5.1 Labeled FACS tubes as shown in the Post-REP TIL            Phenotype worksheet.        -   7.5.2 Added the antibodies as shown in the Post-REP TIL            Phenotype worksheet.        -   7.5.3 After incubation, added 3 mLs of FACS buffer to each            tube.        -   7.5.4 Spun tubes at 500 g, high acceleration and brake, for            2 minutes.        -   7.5.5 Decanted or aspirated the supernatant.        -   7.5.6 Resuspended the tubes with 500 μl of 1% PFA in PBS and            stored at 2-80° C. in the dark.    -   7.6 Data Acquisition        -   7.6.1 Opened FACSDiva software and login.        -   7.6.2 In the cytometer mismatch dialog, clicked “Use CST            Settings”.        -   7.6.3 Created a new experiment by clicking on “Experiment”            tab and selecting the “Extended Phenotype” template.        -   7.6.4 Double clicked on Target Values experiment and            adjusted voltages to reach the target values determined by            flow core operator.        -   7.6.5 Copied instrument settings and pasted them onto the            new experiment.        -   7.6.6 Created a Specimen for each individual and named it            appropriately.        -   7.6.7 Created names for the samples according to the labels            on their tubes.        -   7.6.8 Gently vortexed or flick with finger before placing            the tube in the SIT.        -   7.6.9 Acquired the data under RECORD in the Acquisition            board.        -   7.6.10 Ran the samples at a speed of less than 7,500 events            per second.        -   7.6.11 Collected between 50,000 to 100,000 live events            excluding debris.

Example 19: Process 2a Verification Process Development

The experiments in this Example were completed to analyze Process 2A forthe manufacture of TIL from patient-derived tumors of melanoma and asingle breast cancer including the outgrowth of TIL from tumors in apre-REP procedure, followed by a modified REP. Special emphasis wasplaced on the establishment of a frozen TIL product and a comparison ofthe performance of the frozen TIL product against the current fresh TILproduct process (Process 1C). This report will demonstrate that similarprofiles are observed in assessment of fresh and thawed critical qualityattributes (cell number, % viability, % CD3+ T-cells, andbead-stimulated gamma interferon (IFN-γ) production) as well as are-stimulation extended phenotype procedure (reREP) whether the same TILproduct is fresh or frozen. Data presented to support this conclusioninclude proliferation, viability, phenotype, IFN-γ release, potency,telomere length, and metabolic activity. The results characterize theProcess 2A, a shortened pre-REP/REP process followed by thecryopreservation of TIL as well as compare the 2A process to the longer1C process, as described herein.

Tumor donor descriptions, processing dates and processing locations canbe found in Table 1 below (*indicates that REP was started using afrozen pre-REP TIL line):

TABLE 30 Description of Tumor Donors, Processing Dates And ProcessingLocations. Tumor ID Tissue Type Source Tissue M1061 Melanoma MT groupPrimary - left lateral foot M1062 Melanoma Moffitt N/A M1063 Melanoma MTgroup Metastatic C - right groin M1064 Melanoma MT group Metastatic C -left ankle M1065 Melanoma Bio Metastatic - Axillary Options lymph nodeEP11001 ER+PR+ MT group Primary - left breast invasive ductal carcinomaM1056* Melanoma Moffitt N/A M1058* Melanoma MT group Metastatic - StageIIB Right scalp M1023* Melanoma Atlantic Primary - Right axilla Health

3. Background Information

-   -   3.1 LN-144 is an immunotherapeutic product for treating patients        with metastatic melanoma. The product was composed of autologous        tumor-infiltrating T lymphocytes (TIL) obtained from an        individual patient following surgical resection of a tumor and        expanded ex vivo through cell culture of morcellated tumor        fragments (pre-REP) followed by Rapid Expansion of TIL in the        presence of high dose IL-2, anti-CD3, and co-stimulatory APC.        Following non-myeloablative lympho-depletion preconditioning,        the patient received a single infusion of his/her TIL and        subsequent intravenous infusions of aldesleukin (IL-2) every 8        hours for a maximum of 6 doses. Studies involving alternative        methods of TIL expansion in the setting of Damage Associated        Molecular Pattern Molecules (DAMPs) within the tumor        microenvironment (TNE) have also demonstrated effective        expansion of T-cells useful for therapy (Donia 2014;        Sommerville, 2012).        -   The Process 1C which has been used for commercial production            of TIL involves a production schedule that can take ˜45-55            days to produce an infusible TIL product which is delivered            to an immunodepleted patient within 24 hours. The            immunodepletion of the recipient patient must be timed            precisely with the harvest of the current TIL product.            Delays in harvest or delivery of the fresh product can            negatively impact an immunodepleted patient awaiting            infusion. Process 2A improved upon Process 1C by decreasing            manufacturing lead time and materials, due to the decreased            lengths of both pre-REP and REP procedures. In addition,            Process 2A increased flexibility for product shipment time.            The differences between Process 1C and Process 2A in the            pre-REP, REP and harvest of process (see Table 2) includes:        -   3.1.1 Larger flasks with increased tumor fragment capacity            used in the pre-REP procedure.        -   3.1.2 Steps that made use of closed system or which are            amenable to future adaptation to a closed system.        -   3.1.3 Decreased number of days in both pre-REP and REP            procedures.        -   3.1.4 A direct-to-REP approach, which eliminated the need to            phenotype pre-REP populations prior to selecting specific            populations of pre-REP TIL to proceed to REP.        -   3.1.5 A co-culture with a pre-set number of irradiated,            allogeneic PBMC APC in conjunction with anti-CD3 (clone            OKT3) calculated for sufficient expansion of TIL.        -   3.1.6 An automated cell-washing system for harvest.        -   3.1.7 A CS10-based final formulation that was            cryogenically-preserved prior to shipping.

TABLE 31 Impact of Process 2A on Process 1C. Process Step Process 1CProcess 2A Impact STEP A: After surgery, can be After surgery, can befrozen Same. Obtain Patient frozen after harvest and after harvest andbefore Increased tumor fragments tumor sample before Step B. Step B. perflask STEP B: Physical fragmentation Physical fragmentation Shortenedculture time First Expansion 4 fragments per 10 G- 40 fragments per 1G-REX- Reduced number of steps STEP C: REX-10 flasks 100M flask Amenableto closed system First Expansion 11-21 day duration 11 day duration (3days to Shortened pre-REP-to-REP to Second Growth media medium 14 daysrange) process Expansion comprises IL-2 Growth media medium Reducednumber of steps Transition Step B TILs are frozen comprises IL-2Eliminated phenotyping STEP D: until phenotyped for Step B TILs directlymove selection Second selection then thawed to to Step D on Step B day11 Amenable to closed system Expansion proceed to Step D (~day Step Drequires 25- Reduced number of steps 30) 200 × 10⁶ TIL Shorter REPduration Step D requires >40 × 10⁶ 1 G-REX-500M flask on Closed systemtransfer of TIL Step B day 11 TIL between flasks 6 G-REX-100M flasks25-200 × 10⁶ TIL and 5 × 10⁹ Closed system media on Step D day 0 antigenpresenting cell exchanges 5 × 10⁶ TIL and 5 × 10⁸ feeders on Step B day11 antigen presenting cell Split to ≤6 G-REX-500M feeders per flask onStep flasks on day 16 D day 0 11 day duration for Step D Split to 18-36flasks on Growth media medium Step D day 7 comprises IL-2, OKT-3, 14 dayduration for Step D and antigen-presenting cells Growth media mediumcomprises IL-2, OKT-3, and antigen-presenting cells STEP E: TILharvested via TIL harvested via LOVO Reduced number of steps HarvestTILS centrifugation automated cell washing Automated cell washing STEPF: Fresh product in system Closed system Final HypothermosolCryopreserved product in Reduced loss of product Formulation/ Singleinfusion bag PlasmaLyte-A + 1% HSA during wash Transfer to Limitedshipping stability and CS10 stored in LN2 Shipping flexibility InfusionBag 43-55 days from Step A Multiple aliquots Flexible patient schedulingOverall through Step E Longer shipping stability More timely releasetesting Estimated 22 days from Step A Faster turnaround to patientProcess through Step E Decreased clean room Time throughput DecreasedCost of Goods

4. Abbreviations

-   -   μg microgram    -   μl microliter    -   μm micrometer    -   APC Antigen presenting cells    -   CD Cluster of Differentiation    -   CM Central memory    -   CM1, CM2, Culture Media 1, 2    -   CO2 Carbon dioxide    -   CS10 CryoStor® CS10 cryopreservation medium (BioLife Solutions)    -   Ct PCR threshold cycle    -   DAMPs Damage Associated Molecular Pattern molecules    -   dCt Difference between reference Ct value and test Ct value    -   ddCt Difference between dCt and 10 ng standard Ct value    -   ECAR Extracellular acidification rate (measure of glycolysis)    -   EM Effector memory    -   ER+/PR+ Estrogen Receptor+/Progesterone Receptor+    -   GMP Good Manufacturing Practices    -   HBSS Hanks Balanced Salt Solution    -   HSA Human serum albumin    -   IFN-γ Interferon gamma    -   IL Interleukin    -   IU International units    -   LN2 Liquid nitrogen    -   Ml milliliter    -   Mm millimeter    -   ND Not determined    -   Ng Nanogram    -   ° C. degrees Celsius    -   OCR Oxygen consumption rate (measure of oxidative        phosphorylation)    -   OKT3 Clone designation of anti-CD3 monoclonal antibody    -   PBMC Peripheral Blood Mononuclear Cells    -   PD Process Development    -   REP Rapid Expansion Protocol    -   Rh Recombinant human    -   SOP Standard operating procedure    -   T/S Telomere repeat copy number to single gene copy number ratio    -   TIL Tumor Infiltrating Lymphocyte    -   VDJ Variable, diversity, and joining segments of the T cell        receptor    -   Vα, Vβ The mature T cell receptor variable region segments in        the predominant Tumor Infiltrating Lymphocyte    -   μg microgram    -   μl microliter    -   μm micrometer    -   APC Antigen presenting cells    -   CD Cluster of Differentiation    -   CM Central memory    -   CM1, CM2, Culture Media 1, 2    -   CO2 Carbon dioxide    -   CS10 CryoStor® CS10 cryopreservation medium (BioLife Solutions)    -   Ct PCR threshold cycle    -   DAMPs Damage Associated Molecular Pattern molecules    -   dCt Difference between reference Ct value and test Ct value    -   ddCt Difference between dCt and 10 ng standard Ct value    -   ECAR Extracellular acidification rate (measure of glycolysis)    -   EM Effector memory    -   ER+/PR+ Estrogen Receptor+/Progesterone Receptor+    -   GMP Good Manufacturing Practices    -   HBSS Hanks Balanced Salt Solution    -   HSA Human serum albumin    -   IFN-γ Interferon gamma    -   IL Interleukin    -   IU International units    -   LN2 Liquid nitrogen    -   M1 milliliter    -   Mm millimeter    -   ND Not determined    -   Ng Nanogram    -   ° C. degrees Celsius    -   OCR Oxygen consumption rate (measure of oxidative        phosphorylation)    -   OKT3 Clone designation of anti-CD3 monoclonal antibody    -   PBMC Peripheral Blood Mononuclear Cells    -   PD Process Development    -   REP Rapid Expansion Protocol    -   Rh Recombinant human    -   SOP Standard operating procedure    -   T/S Telomere repeat copy number to single gene copy number ratio    -   TIL Tumor Infiltrating Lymphocyte    -   VDJ Variable, diversity, and joining segments of the T cell        receptor    -   Vα, Vβ The mature T cell receptor variable region segments in        the predominant Tumor Infiltrating Lymphocyte

5. Experimental Design

-   -   5.1 Process 2A        -   5.1.1 Pre-REP: Upon receipt, the tumor was transferred to a            Biological Safety Cabinet (Class II, Type A2). Using sterile            technique, the tumor is removed from the shipping container            and washed in HBSS containing 50 μg/mL gentamicin. The            technician morcellates the tumor into 40×3×3×3 mm fragments            which are transferred to a G-REX-100M flask containing            pre-warmed CM1 media supplemented with 6000 IU/mL rhIL-2.            The flask is placed in a 37° C., 5% CO2 humidified tissue            culture incubator for 11 days. If the tumor generates more            than 40 fragments, then more than one G-REX-100M may be set            up. Cells are then harvested and prepared for the REP.        -   5.1.2 REP: On Day 11, one G-REX-500M flask containing 5 L of            CM2 supplemented with 3000 IU/mL rhIL-2, 30 ng/mL anti-CD3            (Clone OKT3) and 5×109 irradiated allogeneic feeder PBMC            cells is prepared. TIL harvested from the pre-REP G-REX-100M            flask after volume reduction are counted and seeded into the            G-REX-500M flask at a density that can range between 5×10⁶            and 200×10⁶ cells. The flask is then placed in a humidified            37° C., 5% CO₂ tissue culture incubator for five days. On            Day 16, volume of the G-REX-500M flask is reduced, TIL are            counted and their viability determined. At this point, the            TIL are expanded into multiple G-REX-500M flasks (up to a            maximum of six flasks), each with a seeding density of 1×10⁹            TIL/flask. All flasks are then placed in humidified 37° C.,            5% CO₂ tissue culture incubators for an additional six days.            On Day 22, the day of harvest, each flask is volume reduced            by 90%, the cells are pooled together and filtered through a            170 μm blood filter, and then collected into a 3 L Origin            EV3000 bag or equivalent in preparation for automated            washing using the LOVO.        -   5.1.3 Harvest and Final Formulation: TIL are washed using            the LOVO automated cell processing system which replaces            99.99% of cell culture media with a wash buffer consisting            of PlasmaLyte-A supplemented with 1% HSA. The LOVO operates            using spinning filtration membrane technology that recovers            over 92% of the TIL while virtually eliminating residual            tissue culture components, including serum, growth factors,            and cytokines, as well as other debris and particulates.            After completion of the wash, a cell count is performed to            determine the expansion of the TIL and their viability upon            harvest. CS10 is added to the washed TIL at a 1:1            volume:volume ratio to achieve the Process 2A final            formulation. The final formulated product is aliquoted into            cryostorage bags, sealed, and placed in pre-cooled aluminum            cassettes. Cryostorage bags containing TIL are then frozen            using a CryoMed Controlled Rate Freezer (ThermoFisher            Scientific, Waltham, Mass.) according to SOP LAB-018 Rev 000            Operation of Controlled Rate Freezer.    -   5.2 TIL Samples: Four conditions of TIL were collected for        characterization comparison.        -   5.2.1 Fresh harvested TIL (direct from PlasmaLyte-A with 1%            HSA wash buffer) Thawed TIL (direct from thawed final            product bag)        -   5.2.2 Fresh Extended Phenotype reREP TIL (fresh harvested            TIL cultured for 7-14 days with IL-2, PBMC feeders, and            anti-CD3 clone OKT3)        -   5.2.3 Thawed Extended Phenotype TIL (thawed TIL cultured for            7-14 days with IL-2, PBMC feeders, and anti-CD3 clone OKT3)    -   5.3 Testing Overview (See FIG. 2)        -   5.3.1 Pre-REP testing includes evaluating the quantity of            IL-2 and analyzing cell culture metabolites such as glucose,            lactic acid, L-glutamine and ammonia throughout the pre-REP.            -   5.3.1.1 IL-2 quantification: media was periodically                removed from pre-REP culture and tested by ELISA for                IL-2 quantification. Reference R&D Systems Human IL-2                Quantikine ELISA Kit manufacturer's instructions.            -   5.3.1.2 Cell culture metabolite analysis: media was                periodically removed from pre-REP culture and tested for                the following metabolites: glucose, lactic acid,                L-glutamine and ammonia. Reference the Roche Cedex                Bioanalyzer user manual for instructions.        -   5.3.2 REP testing included extended assays such as cell            counts, % viability, flow cytometric analysis of cell            surface molecules, potency (IFN-γ production),            bioluminescent redirected lysis assay, granzyme B            production, cellular metabolism and telomere length            measurement.            -   5.3.2.1 Cell counts and viability: TIL samples were                counted and viability determined using a Cellometer K2                automated cell counter (Nexcelom Bioscience, Lawrence,                Mass.) according to SOP LAB-003 Rev 000 Cellometer K2                Image Cytometer Automatic Cell Counter.            -   5.3.2.2 Flow cytometric analysis of cell surface                biomarkers: TIL samples were aliquoted for flow                cytometric analysis of cell surface markers using the                procedure outlined in WRK LAB-041 Rev 000 Surface                Antigen Staining of Post REP TIL            -   5.3.2.3 Potency Assay (IFN-γ production): Another                measure of cytotoxic potential was measured by                determining the levels of the cytokine IFN-γ in the                media of TIL stimulated with antibodies to CD3, CD28,                and CD137/4-1BB. IFN-γ levels in media from these                stimulated TIL were determined using the WRK LAB-016 Rev                000 Stimulation of TIL to Measure IFN-γ Release            -   5.3.2.4 Bioluminescent Redirected Lysis Assay: The                cytotoxic potential of TIL to lyse target cells was                assessed using a co-culture assay of TIL with the                bioluminescent cell line, P815 (Clone G6), according to                the SOP outlined in WRK LAB-040 Bioluminescent                Redirected Lysis Assay (Potency Assay) for TIL            -   5.3.2.5 Granzyme B Production: Granzyme B is another                measure of the ability of TIL to kill target cells.                Media supernatants restimulated as described in 5.2.5.3                were also evaluated for their levels of Granzyme B using                the Human Granzyme B DuoSet ELISA Kit (R & D Systems,                Minneapolis, Minn.) according to the manufacturer's                instructions.            -   5.3.2.6 Cellular (Respiratory) metabolism: Cells were                treated with inhibitors of mitochondrial respiration and                glycolysis to determine a metabolic profile for the TIL                consisting of the following measures: baseline oxidative                phosphorylation (as measured by OCR), spare respiratory                capacity, baseline glycolytic activity (as measured by                ECAR), and glycolytic reserve. Metabolic profiles were                performed using the procedure outlined in WRK LAB-029                Seahorse Combination Mitochondrial/Glycolysis Stress                Test Assay.            -   5.3.2.7 Telomere length measurement: Diverse methods                have been used to measure the length of telomeres in                genomic DNA and cytological preparations. The telomere                restriction fragment (TRF) analysis is the gold standard                to measure telomere length (de Lange et al., 1990).                However, the major limitation of TRF is the requirement                of a large amount of DNA (1.5{circumflex over ( )}g).                Two widely used techniques for the measurement of                telomere lengths namely, fluorescence in situ                hybridization (FISH; Agilent Technologies, Santa Clara,                Calif.) and quantitative PCR.        -   5.3.3 Additional samples were taken for the following tests,            and could be analyzed in the future as needed:            -   5.3.3.1 In-depth cytokine analysis            -   5.3.3.2 TCR sequencing

6. Results Achieved

A total of 9 experiments were performed using the TIL derived from thetumors described in section 2.3 the experimental design and harvestconditions in section 5.1. TIL harvested using Process 2A were subjectedto the testing outlined in section 5.3.2 for the purpose ofunderstanding their ability to expand, their viability, phenotype,cytotoxic potential, and metabolic profile. All measures were taken forthe fresh harvested TIL product and the thawed frozen TIL product(Process 2A).

-   -   6.1 Cell Counts and Viability        -   6.1.1 Cell counts were taken at the end of the pre-REP, on            Day 5 or 6 of the REP (expansion day), and at the end of the            REP, both prior to LOVO wash and after LOVO wash. The cell            counts were then used to determine the expansion of TIL            during the REP and the recovery of TIL after washing on the            LOVO. After thaw, the cells were counted again to determine            the post-thaw recovery (based on the concentration at which            the TIL were frozen) and the post-thaw viability prior to            proceeding with other analytical assay. Table 3 summarizes            all of these results for the nine Process 2A runs.

TABLE 32 Cell counts, % viability, and expansion of TIL from Process 2Aruns. M1061T M1062T M1063T M1064T M1065T EP11001T M1056T M1058T M1023Tpre-REP 3.3 × 10⁷ 1 × 10⁸ 7.5 × 10⁷ 1.8 × 10⁸ 4.1 × 10⁶ 5.4 × 10⁶ 7 ×10⁷  4.7 × 10⁷ 4.8 × 10⁷ Inoculum Day 5/6 1.3 × 10⁹ 4 × 10⁹   3 × 10⁹3.6 × 10⁹ 6.6 × 10⁸ 2.8 × 10⁹ 4.0 × 10⁹   3.7 × 10⁹ 2.2 × 10⁹ Count Fold898 590  470 130 1900 522  771 1400  850  Expansion from Day 0 to Day 11Harvest  2.8 × 10¹⁰ 5.6 × 10¹⁰   3.5 × 10¹⁰  2.3 × 10¹⁰  7.8 × 10⁹ 2.63× 10¹⁰  5 × 10¹⁰  6.7 × 10¹⁰  4.1 × 10¹⁰ LOVO 100 68 100 100  92 95 10090 99 Recovery (%) Cryostorage 3 × 30 ml    2 × 100 ml 2 × 100 ml 2 × 50ml 3 × 100 ml     2 × 65 ml 2 × 100 ml 2 × 100 ml 2 × 100 ml BagsPost-thaw 103 84  90  88 101 82  82 86 78 Recovery (%) Post-thaw   84.75   84.36    77.15    83.48    79.98   74.85    80.28   85.03  89.21 Viability (%)

-   -   -   6.1.2 Process 2A SOP defines the starting number of TIL for            a REP as a range of 5 200×106 TIL. The range of nine TIL            samples used to start the Process 2A REPs was from 4.1×106            (M1065T)−1.8×108 (M1064T), with an average starting TIL            number of 6.58×107. Interestingly, the REP plated with the            lowest number of TIL expanded to the greatest degree at REP            harvest (range of expansion for all 9 REPs: 130-1900-fold;            average expansion, 840-fold). The average number of TIL            harvested at the end of these nine Process 2A REPs was            4.49×1010 (range 7.8×109−6.7×1010).        -   6.1.3 For comparative statistics of Process 1C, see            Chemistry, Manufacturing, and Controls (CMC) Section of            Investigational New Drug (IND) Application for LN144/LN-145.        -   6.1.4 Process 1C utilizes manual handling and centrifugation            to wash the TIL product. This is time consuming, but more            importantly can result in the loss of up to 25% of the            product between harvest and final formulation. The automatic            cell washing LOVO system provides a way to minimize cell            loss and also introduces a closed system wash which            decreases the risk of contamination of the product during            the wash steps. The recovery of the product following the            LOVO wash step of the protocol and showed an average of            93.8±10.4% recovery of the TIL product going into the wash            step. This statistic includes TIL product for M1062T, which            had a LOVO recovery of 68%, during which an operator error            in the operation of the LOVO resulted in the need to            centrifuge the sample and then restart the LOVO procedure            (see Section 7, Deviations and Discrepancies). This            represents a highly favorable improvement upon the Process            1C washing step on the REP harvest day.        -   6.1.5 Recovery of TIL after the thaw is also a major concern            for a frozen TIL product. Recovery of the product was            determined by measuring the number of cells recovered from            the bag after the thaw compared to the number of cells            placed into each freeze bag prior to cryopreservation. The            range of recovery from thaw was 78-103%, with an average            recovery of 88.2±8.6%.        -   6.1.6 Though there is a significant difference in the            viability of the samples prior to or after thawing, on            average, there is only a 2% loss in viability upon thaw. The            viability of the TIL going into cryopreservation was            84.3±4.7%, and the same TIL after thawing had a viability of            82.1±4.4% (p=0.0742, paired Student's t-test,            non-parametric). Release criteria for the fresh clinical TIL            Process 1C product requires a minimum of 70% viability.            Regardless of a small loss of viability upon thaw, all 9            runs of Process 2A met this release criterion following thaw            of the cryogenic product. Table 4 and FIG. 3 show the            viability of the TIL going into cryopreservation            (Fresh+CS10) and the viability of the TIL upon thaw.

TABLE 33 Comparison of viability of fresh and thawed product. M1061TM1062T M1063T M1064T M1065T EP11001T M1056T M1058T M1023T Fresh + CS1088.05 84.45 82.05 86.75 76.35 77.9 84.8 87.5 90.5 Thaw 84.75 84.36 77.1583.48 79.98 74.85 80.28 85.03 89.21

-   -   6.2 Re-REP expansion of TIL. In addition to examining at the        ability of the fresh product to expand in a REP, the ability of        both the fresh and the thawed TIL product to expand upon        restimulation with fresh irradiated allogeneic PBMC feeder APCs        and fresh anti-CD3 was evaluated. After 7 days, these        restimulated TIL products were analyzed for their ability to        expand from initial culture conditions. FIG. 4 and Table 5 show        the average expansion of re-REP TIL cells after 7 days of growth        in culture. Analysis of the data using a paired Student's t-test        shows that the ability of the TIL to expand in a re-REP is not        significantly different whether starting the REP with a fresh        TIL or thawed TIL product (p=0.81).

TABLE 34 Comparison of fresh and thawed TIL expansion in re-REP cultureM1061T M1062T M1063T M1064T M1065T EP11001T M1056T M1058T M1023T Fresh139.67 264 227 60.12 24.67 268.83 176 316.33 202.33 Thaw 177.33 110.33220.67 177.6 220.2 302.5 114.77 190.67 73.82

-   -   6.3 Cell Culture Metabolites. One of the major premises of Lion        2A was that less tech time and process transfers would lead to        cost savings and limit variability. Possible adverse        consequences of this were increases in undesirable metabolites        and decreases in nutrient sources. As shown in FIG. 5, normal        blood values of electrolytes (sodium and potassium), nutrients        (glutamine and glucose), and metabolites (lactic acid and        ammonia) provide a range to consider when evaluating the results        coming out of the 11 day pre-REP. As shown in FIG. 6, three TIL        (M1061T, M1062T, and M1064T) were evaluated sequentially. In        this setting, potassium and sodium were maintained at normal        levels, glucose was at >1.0 g/L and glutamine>0.3 mmol/L, well        above lower normal blood values. As expected lactate rose to as        high as 0.8 g/L, about 5×the level found in blood normally and        ammonia to as high as 3 mmol/L, as expected from rapidly        expanded cells and also substantially higher than what is found        normally in the blood.    -   6.4 IL-2 Quantification.        -   6.4.1 The main driver of TIL proliferation in the pre-REP in            addition to supplemental glucose, glutamine and sufficient            oxygenation, is the provision of high levels of rhIL-2.            Following its addition to serum containing media, IL-2            levels were measured at 2-3.5×10³ IU/ml, only falling to            about 1.0×10³ IU/ml over the 11 days of culture. This is            well above the 30-100 IU/ml necessary to sustain T-cell            proliferation. Assessment of IL-2 concentrations using            different sources of IL-2 (Prometheus, Akron, Cellgenix) is            currently being tested in separate experiments (QP-17-010:            Qualification of IL-2 from Cellgenix, Akron and Prometheus)            at Lion Biotechnologies, Tampa.    -   6.5 IFN-γ Production        -   6.5.1 After 24 hr stimulation of TIL with magnetic anti-CD3,            CD28 and 4-1BB Dynabeads as described in sections 5.3.5.3,            supernatant from cultures was collected and analyzed for            IFN-γ using ELISA kits. All restimulated TIL produced more            IFN-γ than their unstimulated counterparts, showing that the            stimulation of the TIL resulted in their activation. FIG. 8            shows the ability of the four different TIL compositions            (fresh, thaw, fresh re-REP and thaw re-REP TIL) tested to            release IFN-γ into the surrounding medium upon            restimulation.

Tables 6 and 7 show the average values of IFN-γ secretion in the 9Process 2A runs. IFN-γ secretion into the surrounding medium uponrestimulation is not different between the fresh TIL product and thethawed, cryopreserved TIL. Table 6 shows that fresh product produced anaverage of 4143±2285 pg IFN-γ/106 TIL while thawed product produced3910±1487 pg IFN-γ/106 TIL (p=0.55 using paired Student's t-test). Ifnormalized to total TIL product (Table 7), on average, stimulated freshTIL produced 86±61 grams IFN-γ, while thawed stimulated TIL produced68±40 grams IFN-γ (p=0.13). These findings indicate that both fresh andthawed TIL products produce IFN-γ and that there is no difference in theability of either fresh or thawed matching TIL to produce IFN-γ uponstimulation with anti-CD3/anti-CD28/anti-4-1BB.

TABLE 35 IFN-γ secretion in fresh and thawed TIL (expressed aspg/10⁶cells/24 hrs) M1061T M1062T M1063T M1064T M1065T EP11001T M1056TM1058T M1023T Fresh 4570 3921 5589 619 1363 4263 6065 2983 7918 Thaw3158 3543 5478 1563 2127 5059 4216 4033 6010 Fresh Re-REP 3638 1732 9712676 2753 1461 2374 770 3512 Thaw Re-REP 2970 2060 1273 1074 1744 25225042 4038 923

TABLE 36 IFN-γ secretion in fresh and thawed TIL. All values are in1012(expressed as grams/10⁶cells/24 hrs) M1061T M1062T M1063T M1064TM1065T EP11001T M1056T M1058T M1023T Fresh 67.1 78.4 99.6 8.4 4.8 66.1157.0 109.0 187.0 Thaw 47.7 59.7 87.9 18.7 7.5 64.4 88.9 127.0 111.0

-   -   6.6 Granzyme B Production        -   6.6.1 TIL were stimulated with magnetic anti-CD3, CD28 and            4-1BB Dynabeads for 24 hr as described in 5.2.5.3, and            supernatant from cultures was collected after 24 hr and            analyzed Granzyme B levels by ELISA. All restimulated TIL            produced more Granzyme B than their unstimulated            counterparts, showing that the stimulation of the TIL            resulted in their activation. FIG. 9 shows the ability of            the fresh TIL, fresh re-REP TIL, and thawed re-REP TIL to            release Granzyme B into the surrounding medium upon            restimulation with the cytokine cocktail.

All products showed granzyme B production ranging from 9190 pg/10⁶viable cells to 262000 pg/10⁶ viable cells (Table 8). Table 6 shows thatfresh product produced an average of 60644+42959, while fresh and thawedre-REP produced 93600+67558 and 103878+84515 respectively. Comparisonbetween the fresh re-REP and thawed re-REP showed that there is nodifference in the ability of the TIL obtained from either conditions(p=0.7). Due to the lack of Granzyme B measurement in the thawedproduct, no statistical analysis were performed using the fresh TILproduct.

TABLE 37 Granzyme B secretion in fresh TIL, fresh reREP TIL, and thawedreREP TIL (expressed as pg/10⁶cells/24 hrs) M1061T M1062T M1063T M1064TM1065T EP11001T M1056T M1058T M1023T Fresh 10600 108000 49100 2840024300 17900 120000 12900 79100 Fresh ReREP 216000 37700 42400 91800192000 22200 97300 73800 69200 Thaw ReREP 262000 113000 35100 6560048700 9190 147000 201000 53300

-   -   6.7 Flow cytometric analysis of cell surface biomarkers

Phenotypic profiling of TILs: Four antibody panels have beenstandardized at LION to broadly characterize the functional profile ofT-cells. These panels were used to assess the immunophenotyping of freshTIL, thawed TIL, fresh re-REP TIL, and thawed re-REP TIL. All the dataused for graphical representation in this section are also provided in atable format (Tables 14-24) in the appendix section 10.

-   -   6.8 Bioluminescent Redirected Lysis Assay        -   6.8.1 To determine the potential ability of the Process 2A            TIL to kill their target tumor cells, we developed a potency            assay involving the co-culture of TIL with a bioluminescent            surrogate target cell line P815, as described in section            5.3.2.4. A 4 hour co-culture of the different TIL            compositions with P815 in the presence of anti-CD3            stimulation gives a measure of the cytotoxic potential of            the TIL cells expressed as LU50, lytic units which can be            defined as the number of TIL necessary to kill 50% of the            target cells. This measure is then expressed as LU50/106            TIL. FIG. 32 below shows the cytotoxic potential of the TIL            from the fresh product, and from the two re-REP TIL            conditions, fresh re-REP and thaw re-REP.        -   6.8.2 Comparison of the fresh re-REP to the thaw re-REP            shows that there is no significant difference in the ability            either TIL to kill a target cell (p=0.3126). This data            supports the conclusion that there is no difference between            the fresh and the thawed product in terms of the cytotoxic            potential of the TIL product. No comparison between fresh            was performed as cytotoxic potential was not measured            immediately after thawing TIL. Table 9 shows the lytic units            of TIL needed to kill 50% of the P815 target cell line.

TABLE 38 Lytic units produced by TIL against P815 target cell line FreshFresh reREP Thaw reREP M1061T 21.7 42.3 342 M1062T 5.9 17.0 20.9 M1063T14.2 161 12.5 M10641 22.2 8.7 4.4 M10651 42.6 411 128 8 EP11001T 1.8 4.3147 M1056T 25.0 16.6 18.2 M10513T 76.9 13.8 16.6 M1023T 30.8 25.6 30.4avg ± sd 26.8 ± 22.5 20.6 ± 13.3 31.1 ± 37.6

-   -   6.9 Cellular metabolism profile of TIL        -   6.9.1 To assess the metabolic health of post-REP TIL, we            utilized the Seahorse metabolism analyzer instruments (XFp            and XFe96) from Agilent Technologies (Santa Clara, Calif.)            following the protocol outlined in section 5.3.2.6. Briefly,            by treating cells with inhibitors that target certain            aspects of either oxidative phosphorylation or glycolysis,            cells are stressed in such a way that allows for the            determination of their SRC and glycolytic reserve. In            addition, basal levels of both oxidative phosphorylation            (basal OCR) and glycolysis (basal ECAR) can be determined.            Finally, because inhibitors of oxidative phosphorylation and            glycolysis are combined in the same test, a potential hidden            reserve of SRC can be discerned which is only apparent when            the cells are treated with the competitive inhibitor of            glycolysis, 2-deoxyglucose (2-DG), (labeled SRC2DG),            resulting in an increase in SRC which would otherwise remain            hidden. This extra respiratory capacity has been labeled as            “Covert” SRC. Table 9 shows the metabolic profiles of the            fresh harvested TIL, fresh re-REP TIL, and thawed re-REP TIL            derived from the metabolic stress test performed on the            cells.        -   6.9.2 FIGS. 55A-F show the data from Table 38 in graphical            form. The fresh harvested REP product shows some statistical            differences from the fresh re-REP and thawed re-REP            products. This is not surprising since the re-REP product            has been restimulated with fresh irradiated PBMC APC and            fresh anti-CD3 antibody either immediately after the REP or            upon thaw. However, in all cases, there is no significant            difference between the fresh and thawed products when both            are restimulated in a re-REP procedure (see p values of            Table 9). This indicates that the cryopreservation process            does not detrimentally affect the TIL product. Most notably,            for oxidative phosphorylation, the re-REP products have            higher SRC than their matching fresh harvest REP products.            For glycolysis, the re-REP TIL have statistically            significantly higher basal levels of glycolysis and            conversely statistically lower levels of glycolytic reserve            than fresh REP product. It is worth noting that this could            indicate that the re-REP TIL are more highly activated than            the freshly harvested TIL, as activated, healthy TIL are            reported to possess high levels of glycolytic activity (Buck            et al., JEM 212:1345-1360; 2015).

TABLE 39 Metabolic Profile of Process 2A TIL p v. p v. fresh M1061 M1062M1063 Moff2 Moff3 Moff4 EP11001T M1064 M1065 avg sd fresh re-REP BasalOCR, pmol/min PLLA 50.33 33.95 74.89 36.80 38.48 39.89 63.02 55.89 49.1614.56 fresh re-REP 38.92 38.48 54.35 25.98 18.68 38.61 37.33 41.04 36.6710.57 0.03 thaw re-REP 39.25 43.28 60.05 30.68 57.90 59.08 27.85 52.5832.82 44.83 12.90 0.48 0.11 Overt SRC, pmol/min PLLA 24.74 10.45 101.1847.32 77.00 35.07 31.39 3.02 41.27 33.22 fresh re-REP 51.72 36.46 48.2428.34 37.69 21.02 9.93 99.71 41.64 27.17 0.29 thaw re-REP 47.38 40.40121.86 26.04 37.32 86.47 58.45 89.59 56.45 62.66 30.75 0.16 0.12 SRC20G,pmol/min PLLA 14.01 5.72 35.98 29.97 74.62 24.42 31.39 20.70 29.60 20.67fresh re-REP 81.80 78.82 52.73 38.69 92.37 42.35 −12.81 137.15 63.8944.45 0.08 thaw re-REP 76.97 77.72 177.48 48.27 56.57 69.05 74.14 130.7685.89 88.54 40.59 0.00 0.25 Covert SRC, pmol/min PLLA 0.00 0.00 0.000.00 0.00 0.00 0.00 17.68 2.21 6.25 fresh re-REP 30.08 42.36 4.50 10.3554.68 21.33 0.00 2.63 20.74 20.13 0.02 thaw re-REP 29.59 37.32 55.6222.23 19.25 0.00 15.68 41.16 29.44 27.81 16.10 0.01 0.52 Basal ECAR,mpH/min PLLA 53.44 27.55 136.33 48.72 89.80 62.29 108.38 72.07 74.8235.20 fresh re-REP 96.48 96.63 171.47 102.87 145.19 153.97 35.60 147.02118.65 44.19 0.10 thaw re-REP 143.35 173.93 193.39 149.19 169.21 73.1798.64 96.37 90.55 131.98 43.15 0.01 0.38 Glycolytic Reserve, mpH/minPLLA 32.11 26.18 52.00 19.09 38.01 39.03 43.14 76.43 40.75 17.61 freshre-REP 24.06 8.75 18.17 −8.28 −5.89 10.31 35.34 20.80 12.91 14.85 0.003thaw re-REP 15.50 −18.94 13.56 −6.78 11.45 54.84 −21.37 −12.66 −5.473.35 23.75 0.01 0.47

-   -   -   6.9.3 A direct comparison of fresh to frozen products using            the re-REP procedure has enabled us to determine that both            the fresh and frozen TIL products, upon identical            stimulation conditions, result in metabolic profiles that            are statistically indistinct. Both fresh re-REP and thawed            re-REP TIL have similar levels of basal respiration (FIG.            60A, 36.7±10.6 and 44.8±12.9 pmol/min, respectively; p=0.11)            as well as similar (overt) SRC (FIG. 60B, 41.6±27.2 and            62.7±30.8; p=0.12). Upon treatment of these re-REP cells            with 2-DG, the competitive inhibitor to glucose, which            results in an inhibition of glycolysis, we see that both            fresh and thawed re-REP TIL show an extra, “hidden” spare            respiratory capacity (SRC2DG; Covert SRC) that is mostly low            or absent in the fresh harvested TIL sample (FIG. 60C); only            one sample had high levels of SRC2DG (FIG. 60C) in the fresh            harvested TIL, while conversely, only one of seven samples            tested showed a lack Covert SRC upon re-REP. Covert SRC            (FIG. 60D) for fresh re-REP averaged 20.7±20.1 while covert            SRC (FIG. 60D) for thawed re-REP ranged from 27.8±16.1;            p=0.52).        -   6.9.4 The most striking metabolic readout of the extended            phenotype (re-REP) TIL is the consistently high levels of            basal glycolysis of the extended phenotype (re-REP) samples.            Basal glycolysis (FIG. 60E) is consistently high in re-REP            samples, averaging 118.7±44.2 mpH/min in the fresh re-REP            and 132.0±43.2 mpH/min in the thawed re-REP. These samples            are not statistically different from each other (p=0.38).            However, as mentioned above, the fresh harvested sample does            not possess such high basal levels of glycolysis. Compared            to fresh re-REP TIL, this difference is substantial, but not            significant (p=0.10); however when compared to the thawed            re-REP samples, the difference is significant (p 0.01).            These re-REP cells are apparently heavily reliant on            glycolysis for their energy needs, as they have little            glycolytic reserve remaining when stressed in the Seahorse            metabolic tests (FIG. 60F): fresh re-REP TIL average            12.9±14.9 mpH/min; thawed re-REP TIL, 3.35±23.8 mpH/min).            These re-REPs are not different from each other (p=0.47) but            both are statistically different than the glycolytic reserve            found in fresh harvested TIL samples, which averages            40.8±17.6 mpH/min (p=0.003 and 0.01 compared to fresh re-REP            and thawed re-REP TIL, respectively). Further studies should            be conducted to determine the cause behind the differences            seen in glycolysis between these fresh harvest and re-REP            TIL samples.

    -   6.10 Telomere Length Measurement        -   6.10.1 Measurement of Telomere Length of Post REP TIL by            Flow Fish and qPCR.            -   6.10.1.1 Flow-FISH was performed using Dako/Agilent                Pathology Solutions (Telomere PNA Kit/FITC for Flow                Cytometry) kit and the manufacturer's instructions were                followed to measure the average length of the Telomere                repeat. 1301 T-cell leukemia cell line (Sigma-Aldrich,                St. Louis, Mo.)) was used as an internal reference                standard in each assay. Individual TIL were counted and                mixed with 1301 cells at a 1:1 cell ratio. 2×106 TIL                were mixed with 2×106 1301 cells. In situ hybridization                was performed in hybridization solution (70% formamide,                1% BSA, 20 mM Tris, pH 7.0) in duplicate and in the                presence and absence of a FITC-conjugated Telomere PNA                probe (FITC-00-CCCTAA-CCC-TAA-CCC-TAA) complementary to                the telomere repeat sequence at a final concentration of                60 nM. After addition of the Telomere PNA probe, cells                were incubated for 10 minutes at 82° C. in a heat block.                The cells were then placed in the dark at room                temperature overnight. The next morning, excess telomere                probe was removed by washing 2 times for 10 minutes each                on a heat block at 40° C. with Wash Solution. Following                the washes, DAPI (Invitrogen, Carlsbad, Calif.) was                added at a final concentration of 75 ng/ml. DNA staining                with DAPI was used to gate cells in the G0/G1                population. Sample analysis was performed using a Yeti                flow cytometer (Propel-Labs, Fort Collins, Colo.).                Telomere fluorescence of the test sample was expressed                as a percentage of the fluorescence (fl) of the 1301                cells per the following formula:Relative telomere                length=[(mean FITC fl test cells w/probe-mean FITC fl                test cells w/o probe)×DNA index of 1301                cells×100]/[(mean FITC fl 1301 cells w/probe−mean FITC                fl 1301 cells w/o probe)×DNA index of test cells.            -   6.10.1.2 qPCR: Real time qPCR was used to measure                relative telomere length. Briefly, the telomere repeat                copy number to single gene copy number (T/S) ratio was                determined using an Bio-Rad PCR thermal cycler (Bio-Rad                Laboratories, Hercules, Calif.) in a 96-well format. Ten                nanograms of genomic DNA was used for either telomere                (Tel) or hemoglobin (hgb) PCR reaction and the primers                used were as follows: Tel-1b primer (CGG TTT GTT TGG GTT                TGG GTT TGG GTT TGG GTT TGG GTT), Tel-2b primer (GGC TTG                CCT TAC CCT TAC CCT TAC CCT TAC CCT TAC CCT), hgb1                primer (GCTTCTGACACAACTGTGTTCACTAGC), and hgb2 primer                (CACCAACTTCATCCACGTTCACC). All samples were analyzed by                both the telomere and hemoglobin reactions, and the                analysis was performed in triplicate on the same plate.                In addition to the test samples, each 96-well plate                contained a five-point standard curve from 0.08 ng to                250 ng using genomic DNA isolated from 1301 cells. The                T/S ratio (−dCt) for each sample was calculated by                subtracting the median hemoglobin threshold cycle (Ct)                value from the median telomere Ct value. The relative                T/S ratio (−ddCt) was determined by subtracting the T/S                ratio of the 10 ng standard curve point from the T/S                ratio of each unknown sample.            -   6.10.1.3 Telomere Length Results and Discussion:                Telomeres are caps (repetitive nucleotide sequences) at                the end of the linear chromosomes which play a critical                role in facilitating complete chromosome replication                Telomere measurement is an emerging tool in the study of                such conditions as degenerative diseases, cancer, and                aging. Previous studies from NIH (J Immunol. 2005, Nov.                15; 175(10):7046-52; Clin Cancer Res. 2011, Jul. 1;                17(13): 4550-4557) have shown that longer telomere                length of TIL is associated with clinical response.                Conversely, Radvanyi's group found no significant                difference in the telomere length of TIL between                responders and non-responders (Clin Cancer Res; 18(24);                6758-70). Thus far, there is no evidence to prove that                telomere length is associated with the length of in                vitro T cell culture. It is possible that post-REP TIL                cultured by Process 2A (22 day culture) will have longer                telomere length when compared to TIL cultured by Process                1C process (25-36 day culture).

7. Discrepancies and Deviations

-   -   7.1 Process Deviations        -   7.1.1 M1061T: REP cells were split on Day 6 into 4 G-Rex500M            flasks.        -   7.1.2 M1062T: REP cells were split on Day 6 into 4 G-Rex500M            flasks. Due to an operator error on the LOVO filtration            system, an emergency stop occurred during the procedure            which required a manual collection of the TIL from the            disposable kit. The TIL were successfully filtered during a            second LOVO run.        -   7.1.3 M1063T: No deviations M1064T: No deviations        -   7.1.4 M1065T: Pre-REP cells were below specification for            cell count on Day 11 (<5×106 cells) but were continued into            the REP. On REP Day 6, the cells were counted and placed            back into the G-Rex500M and fed with 4.5 L fresh media. The            TIL were not expanded on this day due to insufficient cell            count (<1×109 cells on REP Day 6).        -   7.1.5 EP11001T: No deviations        -   7.1.6 M1056T: Pre-REP cells were cultured at LION in a G-Rex            100 flask for up to 21 days. Tumor fragments were filtered            out on pre-REP Day 11 and the TIL were frozen down on day of            harvest in 100% CS10 at 30×106 cells per 1.5 ml vial. Frozen            TIL were thawed at Moffitt PD in CM1 supplemented with 6000            IU/mL rhIL-2 and rested for 3 days before initiating Day 0            of the REP. On REP Day 6, TIL were expanded into 4 flasks            which proceeded to harvest on REP Day 11.        -   7.1.7 M1058T: Pre-REP cells were cultured at LION in a G-Rex            100 flask for up to 21 days. Tumor fragments were filtered            out on pre-REP Day 11 and the TIL were frozen down on day of            harvest in 100% CS10 at 30×106 cells per 1.5 ml vial. Frozen            TIL were thawed at Moffitt PD in CM1 supplemented with 6000            IU/mL rhIL-2 and rested for 3 days before initiating Day 0            of the REP. On REP Day 6, cells were split into 4 flasks            which proceeded to harvest on REP Day 11.        -   7.1.8 M1023T: Pre-REP cells were cultured at LION in G-Rex10            flasks for up to 21 days. Tumor fragments were filtered out            on pre-REP Day 11 and the TIL were frozen down on day of            harvest in 100% CS10 at 30×106 cells per 1.5 ml vial. Frozen            TIL were thawed at Moffitt PD in CM1 supplemented with 6000            IU/mL rhIL-2 and rested for 3 days prior to initiating Day 0            of the REP. On REP Day 6, cells were expanded into 4 flasks            which proceeded to harvest on REP Day 11.    -   7.2 Testing Deviations        -   7.2.1 In-depth cytokine analysis and TCR sequencing were not            performed

8. Conclusions and Recommendations

-   -   8.1 Developing a More Robust Process. The challenge to Lion was        to convert the earlier Lion Process 1C, which had a long        processing time, to a potentially more commercializable Lion        Process 2A which utilizes refinements resulting in shorter        processing time and a cryopreserved final formulation of the TIL        product. To this end, nine Process Development runs were        conducted to confirm that the old and new processes demonstrated        comparable cell yields and comparable TIL potency and phenotype.        Of particular note was the markedly decreased complexity of the        overall process, resulting in a 50% reduction in the overall        length of the pre-REP and REP processes, yet still resulting in        comparable TIL yields (7.8×109−67×109 cells) compared to the        historic Lion Process 1C currently practiced at our contract        manufacturer. This was recently updated for the June 2017 ASCO        presentation (Mean: 41.04×109 cells with a range of 1.2-96×109        cells). In addition, Lion has successfully developed a        cryopreserved TIL product which demonstrated a post-thaw        recovery of 78-103% with >70% viability of TIL, consistent with        current Process 1C release criteria (see Table 2).    -   8.2 The Role of the Extended Phenotypic Analysis (Re-REP). The        ability to proliferate in response to mitogenic stimulation (as        in the experimental re-REPs presented in this report) is a        critical quality attribute of TIL. The experiments presented        here show that 8/9 thawed TIL products were able to        expand >100-fold in one week compared to 7/9 matched fresh TIL        products, supporting the comparability of the thawed TIL product        to the fresh TIL product (Table 2). Two additional critical        quality attributes of TIL are their ability to release IFN-γ        and/or Granzyme B following cytokine (CD3/CD28/4-1BB)        stimulation. Cytokine stimulation of both the fresh and thawed        products resulted in IFN-γ release exceeding 2 ng/10⁶ cells/24        hours in 7/9 fresh products and all thawed products (FIG. 35)        (see section 6.2 of this report). Granzyme B release (FIG. 36)        was observed in all 9 process runs. CD4 and CD8 levels (FIG. 39        and FIG. 40) demonstrated remarkable internal consistency        between fresh and thawed TIL products. In addition, analysis of        the ability of the TIL to kill a surrogate tumor target cell        line (P815, FIG. 59) showed that the fresh and thawed TIL        possessed similar cytotoxic potential.    -   8.3 A Metabolic Stress Test of TIL Reveals Robust Bioenergetics.        An analysis of the metabolic profiles of fresh and thawed TIL        products stimulated in a re-REP demonstrated that both fresh and        thawed TIL responded similarly to metabolic stress testing and        showed no substantive differences in a panel of metabolic        characteristics (Table 39). Thus, the cryopreserved Process 2A        TIL product can be considered comparable to the fresh Process 1C        product based on the four quality attributes of identity,        potency, cell number, and viability presented in this report.        Assays comparing matched fresh and thawed cells were quite        comparable in every assay outlined in this report.    -   8.4 Acceptance Criteria: The intrinsic heterogeneity of TIL        products with personalized therapy for each patient        reflects: (1) their unique major histocompatibility complex        restricting molecules (the most polymorphic gene products in        human biology); (2) the unique evolutionary trajectory of        individual tumors arising in the tumor microenvironment with        genomic instability and unique individual driver and passenger        mutations; and (3) the heterogeneity conferred by allelic        variation, N-region diversity, and VDJ rearrangements in the Vα        and Vβ segments defining the T-cell receptors used for        recognition of neoepitopes shared tumor-testis antigens, and        virally encoded products. Assessing additional variation        occurring as the result of process changes is thus a daunting        task and requires assessment of as many parameters as possible        to assure oneself that ‘comparability’ of an intrinsically        heterogeneous material as possible. This has been accomplished        by faithfully examining several acceptance criteria for        feasibility and comparability as detailed in the Table 40 below.

TABLE 40 Acceptance criteria for feasibility and comparabilityAcceptance Criteria for Acceptance Criteria Sampling Point ParameterTest Method Feasibility for Comparability Total Viable Cells AutomatedCell ≥1.5 × 10⁹ viable cells No statistical Counter with AOPIsignificance between fresh and frozen ReREP arms (p- value<0.05) %Viability Automated Cell ≥70% viable No statistical Counter with AOPIsignificance between fresh and frozen ReREP arms (p- value<0.05) PurityFlow Cytometry ≥90% T-cells No statistical significance between freshand frozen ReREP arms (p- value<0.05) TCR Sequencing N/A N/A PotencyIFNI(ELISA ≥2x background No statistical and significance between ≥400pg/1 × 10⁶ viable cells/24 hrs fresh and frozen ReREP arms (p-value<0.05) Granzyme B ELISA ≥2x background N/A Bioluminescent N/A N/ARedirected Lysis Assay Respiration Seahorse Stress N/A N/A Test

Based on the feasibility criteria listed in Table 11, TIL will beevaluated on whether or not the requirements were met. All individualcriteria were met for each experiment and each TIL line (n=9). Studentt-test was used for statistical analysis. Non-parametric student T-testwas used to calculate the p-value for % viability as viability measureswill not be a Gaussian distribution. See, Table 41 below.

TABLE 41 Meeting Feasibility Acceptance Criteria. Potency (IFNy Purity(Flow ELISA) Cell Count % Viability Cytometry) pg/1 ×10⁶cells/24h TILLine Fresh Thaw Fresh Thaw Fresh Thaw Fresh Thaw M1061T 6.48 × 10⁹ 6.66× 10⁹ 88.05 84.93 95.3 91.5 4570 3158 M1062T 6.76 × 10⁹ 5.70 × 10⁹ 84.4583.73 99.7 98.9 3921 3543 M1063T 14.9 × 10⁹ 13.5 × 10⁹ 82.05 77.15 98.799.6 5589 5478 M1064T 8.06 × 10⁹ 7.08 × 10⁹ 86.75 83.36 84.5 89.8 6191563 M1065T 3.06 × 10⁹ 3.10 × 10⁹ 76.35 80.90 96.8 91.4 1363 2127EP11001T 14.9 × 10⁹ 12.2 × 10⁹ 77.9 74.85 90.4 94.3 4263 5059 M1056T13.1 × 10⁹ 10.7 × 10⁹ 84.8 80.20 94.2 94.1 6065 4216 M1058T 23.4 × 10⁹20.1 × 10⁹ 87.5 85.07 99 96.2 2983 4033 M10231 18.4 × 10⁹  144 × 10⁹90.5 89.52 96.5 98.8 7918 6010 P value 0.1132 0.0742 0.9855 0.5821Significantly No No No No different

Based on the acceptance criteria listed in Table 40, fresh and frozenre-REP TIL were evaluated on whether or not the requirements were met.(Viability not reported since the duration of re-REP was 7 days andresidual irradiated PBMC could not be distinguished from TIL.) Numbersin parentheses denote the criteria that were not met. Based on thepurity criteria measured using CD3+ expression, 6/9 fresh Re-REP TILproducts met the stringent >90% criteria (M1061, M1065 and EP11001 didnot) and 8/9 thawed products passed the acceptance criteria evenfollowing Re-REP. The low number of CD3+ TIL in EP11001T fresh re-REPmight be attributed to extreme downregulation of T cell receptor.Measurement of CD3+ TIL as a measure of purity was not determined forM1023T thaw re-REP TIL. For this TIL composition, purity was estimatedusing TCRap staining and is denoted by an asterisk (*). Student-t testwas used for the statistical analysis. See, Table 42 below.

TABLE 42 Meeting Comparability Acceptance Criteria. Potency (IFNy ELISA)Purity (Flow pg/1 × Cell Count Cytometry) 10⁶cells/24h Fresh Thaw FreshThaw Fresh Thaw Re- Re- Re- Re- Re- Re- TIL Line REP REP REP REP REP REPM1061T 1.40 × 10⁶  1.77 × 10⁶ (86.1) 99.3 3638 2970 M10621 2.64 × 10⁶ 1.10 × 10⁶ 99.3 97.1 1732 2060 M1063T 2.27 × 10⁶  2.21 × 10⁶ 99.2 97.4971 1273 M10641 1.76 × 10⁶  1.15 × 10⁶ 83.8 37.8 2676 1074 M1065T 3.16 ×10⁶  1.91 × 10⁶ (78.1) (75.8) 2753 1744 EP11001T 2.02 × 10⁶  0.738 ×10⁶   (18.2) 85.4 1461 2522 M10561 0.601 × 10⁶   1.78 × 10⁶ 98.1 96.72374 5042 M1058T 0.740 × 10⁶   2.20 × 10⁶ 98.4 99.2 770 4038 M1023T 2.69× 10⁶  3.03 × 10⁶ 97 39.9* 3512 923 P value 0.6815 0.3369 0.7680 Sig- NoNo No nificantly different

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9. Additional Tables

TABLE 43 FIG. 39: CD4+ cells Tumor Id M1061 M1062 M1063 M1064 M1065EP11001 M1056 M1058 M1023 Fresh 4.85 34 10.5 41.7 64.9 64.7 4.15 12.38.38 Thaw 5.68 33 11.3 49.5 61.7 62.6 3.46 17.9 7.6 Fresh ReREP 8.1 23.519.2 39/ 31.9 16.3 6.46 12.9 16.7 Thaw ReREP 11 33 15.3 49.3 39.3 26.79.51 17.2 19.1

TABLE 44 FIG. 40: CD8+ cells Tumor ID M1061 M1062 M1063 M1064 M1065EP11001 M1056 M1058 M1023 Fresh 45.6 54.7 85.8 38.2 28.6 22.3 93.2 8488.8 Thaw 50.8 55.7 76.7 37 22.8 19 92.9 76.6 84.3 Fresh ReREP 63 48.372.4 37.9 47.8 5.87 90.3 74.5 74.4 Thaw ReREP 66.3 46.7 47 21.6 19.19.23 82.8 63.7 64.3

TABLE 45 FIG. 41: CD4+CD154+ cells and FIG. 105: CD8+CD154+ cells M1061M1062 M1063 M1064 M1065 T cell Markers Fresh Thawed Fresh Thawed FreshThawed Fresh Thawed Fresh Thawed CD4 C0154+ 78.6 nd nd nd 93.3 62.1 9476.2 91 87.2 CD8 CD154⁺ 37.3 nd nd nd 85.8 19.9 89.3 61.1 17 20.3EP11001 M1056 M1058 M1023 T cell Fresh Thawed Fresh Thawed Fresh ThawedFresh Thawed CD4 79.1 83.1 89.3 92 90.1 92.6 77.9 66.9 CD8 40 36.9 2327.6 40.5 52.1 17.9 13.7 M1061 M1062 M1063 M1064 M1065 Fresh ThawedFresh Thawed Fresh Thawed Fresh Thawed Fresh Thawed T cell MarkersRe-REP Re-REP Re-REP Re-REP Re-REP Re-REP Re-REP Re-REP Re-REP Re-REPCD4 CD154+ 88.9 84 56 82.1 68.2 93.6 97 90.3 0 91.6 CD8 CD154+ 35.6 4912.5 19 59.1 77.88 0.025 90.1 0.00609 61.8 EP11001 M1056 M1058 M1023Fresh Thawed Fresh Thawed Fresh Thawed Fresh Thawed T cell Re-REP Re-REPRe-REP Re-REP Re-REP Re-REP Re-REP Re-REP CD4 52.1 87.1 77 86.4 92.785.1 90.7 81.3 CD8 45.3 74.8 47.3 81.7 73.6 78.3 24.2 27.1

TABLE 46 FIG. 43: CD4+CD69+ cells and FIG. 17: CD8+CD69+ cells M1061M1062 M1063 M1064 M1065 T cell Markers Fresh Thawed Fresh Thawed FreshThawed Fresh Thawed Fresh Thawed CD4 CD69+ 33.9 nd nd nd 82.2 68.8 51.384.8 82.7 84.4 CD8 CD69+ 22.4 nd nd nd 83 78.3 67.8 78.6 78.9 72.3EP11001 M1056 M1058 M1023 T cell Fresh Thawed Fresh Thawed Fresh ThawedFresh Thawed CD4 78.7 58.3 83.9 84.9 89.7 644.6 33.8 38.7 CD8 69.5 54.580.3 86 68 77.8 41.3 48.8 M1061 M1062 M1063 M1064 M1065 Fresh ThawedFresh Thawed Fresh Thawed Fresh Thawed Fresh Thawed T cell MarkersRe-REP Re-REP Re-REP Re-REP Re-REP Re-REP Re-REP Re-REP Re-REP Re-REPCD4 CD69+ 58.7 69.6 67.6 77.6 77.6 86.7 85.5 78.5 90.2 93.2 CD8 CD69+80.9 80 62.7 73.2 87.6 87.9 92.2 88.3 91.3 90.5 EP11001 M1056 M1058M1023 Fresh Thawed Fresh Thawed Fresh Thawed Fresh Thawed T cell Re-REPRe-REP Re-REP Re-REP Re-REP Re-REP Re-REP Re-REP CD4 74.7 39.1 96.1 93.891.1 93.7 35.3 80.1 CD8 87.6 52.9 95.4 94.2 93.1 93.6 71.1 88.1

TABLE 47 FIG. 45: CD4+CD137+ cells and FIG. 19 CD8+CD137+ cells M1061M1062 M1063 M1064 M1065 T cell Markers Fresh Thawed Fresh Tha_(w)edFresh Thawed Fresh Thawed Fresh Thawed CD4 CD137+ 19.8 nd nd nd 65.430.4 nd 1.31 524 7.26 CD8 CD137+ 19.8 nd nd nd 65.4 30.4 nd 1.31 3.237.26 EP11001 M1056 M1058 M1023 T cell Fresh Thawed Fresh Thawed FreshThawed Fresh Thawed CD4 7.78 5.4 4.28 3.65 6.89 4.6 4.28 9.67 CD8 7.785.4 4.28 3.65 6.89 4.6 4.28 9.67 M1061 M1062 M1063 M1064 M1065 FreshThawed Fresh Thawed Fresh Thawed Fresh Thawed Fresh Thawed T cellMarkers Re-REP Re-REP Re-REP Re-REP Re-REP Re-REP Re-REP Re-REP Re-REPRe-REP CD4 CD137+ 15.4 30.4 73 78.1 62.6 53.2 51.6 64.7 31.1 24.6 CD8CD137+ 28.8 43.1 39.3 35.3 84.4 85.7 71.1 81 50.9 33.8 EP11001 M1056M1058 M1023 Fresh Thawed Fresh Thawed Fresh Thawed Fresh Thawed T cellRe-REP Re-REP Re-REP Re-REP Re-REP Re-REP Re-REP Re-REP CD4 65.1 47.8221 18.6 61.6 56.9 49.8 50.8 CD8 57.3 54.6 77.3 78.8 76.9 87 58 50.3

TABLE 48 FIG. 47: CD4+CM cells and FIG. 21 CD8+CM cells M1061 M1062M1063 M1064 M1065 T cell Markers Fresh Thawed Fresh Thawed Fresh ThawedFresh Thawed Fresh Thawed CD4 CM 1.08 n/d 0.59 0.29 10.4 2.08 14.4 0.130.42 0.53 CD8 CM 0.37 n/d 0.9 0.17 3.2 0.66 73.2 0.13 0.21 0.67 EP11001M1056 M1058 M1023 T cell Fresh Thawed Fresh Thawed Fresh Thawed FreshThawed CD4 0.48 1.17 1.83 1.5 1.36 1.8 2.45 1.79 CD8 2.65 1.79 0.33 0.720.91 0.67 1.99 2.22 M1061 M1062 M1063 M1064 M1065 Fresh Thawed FreshThawed Fresh Thawed Fresh Thawed Fresh Thawed T cell Markers Re-REPRe-REP Re-REP Re-REP Re-REP Re-REP Re-REP Re-REP Re-REP Re-REP CD4 CM2.32 7.71 13.8 12.6 13.4 22.3 15.9 18.6 7.03 2.28 CD8 CM 1.85 9.38 6.4814.2 15.7 25.7 24.2 25.8 5.05 1.6 EP11001 M1056 M1058 M1023 Fresh ThawedFresh Thawed Fresh Thawed Fresh Thawed T cell Re-REP Re-REP Re-REPRe-REP Re-REP Re-REP Re-REP Re-REP CD4 18.9 3.73 49.6 55.6 20.1 12.622.1 12.7 CD8 11.4 3.37 25.8 26.4 21.6 19.8 11.1 7.59

TABLE 49 FIG. 49: CD4+EM cells and FIG. 23 CD8+EM cells M1061 M1062M1063 M1064 M1065 T cell Markers Fresh Thawed Fresh Thawed Fresh ThawedFresh Thawed Fresh Thawed CD4 EM 90 n/d 98.3 98.9 83.9 97.2 84.1 99.899.4 99.4 CD8 EM 89.1 n/d 80.6 87.9 92.4 97.8 20.8 98.8 98.3 98.6EP11001 M1056 M1058 M1023 T cell Fresh Thawed Fresh Thawed Fresh ThawedFresh Thawed CD4 96.7 97.4 97.1 97.8 97.4 97.6 9.62 95.3 CD8 91.8 95.598.8 98.9 98.8 99.2 93.9 95.2 M1061 M1062 M1063 M1064 M1065 Fresh ThawedFresh Thawed Fresh Thawed Fresh Thawed Fresh Thawed T cell MarkersRe-REP Re-REP Re-REP Re-REP Re-REP Re-REP Re-REP Re-REP Re-REP Re-REPCD4 EM 95.6 84.4 84.5 83.4 84.3 73.7 80.6 80.4 91.7 97 CD8 EM 97.2 87.990.8 82.3 82.5 72.2 74.5 73 91.5 96.1 EP11001 M1056 M1058 M1023 FreshThawed Fresh Thawed Fresh Thawed Fresh Thawed T cell Re-REP Re-REPRe-REP Re-REP Re-REP Re-REP Re-REP Re-REP CD4 74.3 90.7 36.2 25.5 73.981.8 73.1 76.4 CD8 83 90.8 73.2 71.9 77.1 78.2 84.1 85.1

TABLE 50 FIG. 51: CD4+CD28+ cells and FIG. 25 CD8+CD28+ cells M1061M1062 M1063 M1064 M1065 T cell Markers Fresh Thawed Fresh Thawed FreshThawed Fresh Thawed Fresh Thawed CD4 CD28+ 4.6 5.85 33.2 37 10.5 11.231.9 27.6 41.7 38.2 CD8 CD28+ 30.1 34 24.5 23.1 83.8 49.3 22.5 15.5 13.48.52 EP11001 M1056 M1058 M1023 T cell Fresh Thawed Fresh Thawed FreshThawed Fresh Thawed CD4 63.2 59.8 3.97 3.29 12.2 17.5 8.27 7.48 CD8 14.512 53 54.4 56.5 62.1 76.5 80.8 M1061 M1062 M1063 M1064 M1065 FreshThawed Fresh Thawed Fresh Thawed Fresh Thawed Fresh Thawed T cellMarkers Re-REP Re-REP Re-REP Re-REP Re-REP Re-REP Re-REP Re-REP Re-REPRe-REP CD4 CD28+ 6.75 7.18 21.6 27.8 18.6 15 23 27.6 12.3 15.2 CD8 CD28+24.6 17.9 10 6.4 28.6 18.9 15.7 11 6.9 2.43 EP11001 M1056 M1058 M1023Fresh Thawed Fresh Thawed Fresh Thawed Fresh Thawed T cell Re-REP Re-REPRe-REP Re-REP Re-REP Re-REP Re-REP Re-REP CD4 13.3 20 6.22 9.29 12.316.5 15.4 17.9 CD8 2.07 3.75 24 34 27 36.9 42 43.9

TABLE 51 FIG. 53: CD4+PD-1+ cells and FIG. 27 CD8+PD-1+ cells M1061M1062 M1063 M1064 M1065 T cell Markers Fresh Thawed Fresh Thawed FreshThawed Fresh Thawed Fresh Thawed CD4 PD-1+ 48.5 nd nd nd 77 40.6 nd 22.47.87 7.23 CD8 PD-1+ 37.1 nd nd nd 56 24.6 nd 14 1.61 0.72 EP11001 M1056M1058 M1023 T cell Fresh Thawed Fresh Thawed Fresh Thawed Fresh ThawedCD4 33.3 28.2 33.9 32.8 41.7 38 22.7 23.8 CD8 19.2 12.5 23.8 24.7 78.459.8 42.6 36.1 M1061 M1062 M1063 M1064 M1065 Fresh Thawed Fresh ThawedFresh Thawed Fresh Thawed Fresh Thawed T cell Markers Re-REP Re-REPRe-REP Re-REP Re-REP Re-REP Re-REP Re-REP Re-REP Re-REP CD4 PD-1+ 36.834.2 15.7 26.7 43.9 66 32.4 14.5 22.4 15.5 CD8 PD-1+ 40.4 35.3 6.3 6.2118 20.4 35.6 23.2 6.49 5.73 EP11001 M1056 M1058 M1023 Fresh Thawed FreshThawed Fresh Thawed Fresh Thawed T cell Re-REP Re-REP Re-REP Re-REPRe-REP Re-REP Re-REP Re-REP CD4 40.9 33.4 56 51.3 40.3 32.5 18.9 27.3CD8 29.8 34.6 18.9 15.2 68.6 47 28.9 36.1

TABLE 52 FIG. 55: CD4+LAG3+ cells and FIG. 29 CD8+LAG3+ cells M1061M1062 M1063 M1064 M1065 T cell Markers Fresh Thawed Fresh Thawed FreshThawed Fresh Thawed Fresh Thawed CD4 LAG3+ 16.8 nd nd nd 93.5 37.3 nd6.8 47.2 30.5 CD8 LAG3+ 74 nd nd nd 98.4 81.5 nd 31.8 85.3 38.7 EP11001M1056 M1058 M1023 T cell Fresh Thawed Fresh Thawed Fresh Thawed FreshThawed CD4 35.5 20.1 25 27.4 48.6 38 14.5 7.65 CD8 89.6 64.2 83.4 81.993.2 66.3 90.3 71.1 M1061 M1062 M1063 M1064 M1065 Fresh Thawed FreshThawed Fresh Thawed Fresh Thawed Fresh Thawed T cell Markers Re. REPRe-REP Re. REP Re-REP Re. REP Re-REP Re. REP Re-REP Re. REP Re-REP CD4LAG3+ 68.3 73.1 35.2 56.9 26.9 27.3 52.6 64 65.8 68.2 CD8 LAG3+ 98.398.7 97.1 97.7 89.6 85.1 92.8 94.7 95.4 97.8 EP11001 M1056 M1058 M1023Fresh Thawed Fresh Thawed Fresh Thawed Fresh Thawed T cell Re. REPRe-REP Re. REP Re-REP Re. REP Re-REP Re. REP Re-REP CD4 40.9 46 44.139.1 52.1 51 48.5 17.7 CD8 92.4 92.5 97.5 98.4 98.2 98.3 97.7 78.1

TABLE 53 FIG. 57: CD4+TIM-3+ cells and FIG. 31 CD8+TIM-3+ cells M1061M1062 M1063 M1064 M1065 T cell Markers Fresh Thawed Fresh Thawed FreshThawed Fresh Thawed Fresh Thawed CD4 Tim3+ 89.7 nd nd nd 98.3 87.6 nd43.2 95 78.8 CD8 Tim3+ 99 nd nd nd 99.4 88.1 nd 47 96.9 50.6 EP11001M1056 M1058 M1023 T cell Fresh Thawed Fresh Thawed Fresh Thawed FreshThawed CD4 96.9 91.5 96.4 92.5 88.7 80.1 89.9 82.3 CD8 98.8 83 98.3 92.996.5 73.6 98.2 88.5 M1061 M1062 M1063 M1064 M1065 Fresh Thawed FreshThawed Fresh Thawed Fresh Thawed Fresh Thawed T cell Markers Re-REPRe-REP Re-REP Re-REP Re-REP Re-REP Re-REP Re-REP Re-REP Re-REP CD4 Tim3+95.3 98 94.5 96.9 90.8 90.2 94.2 82.6 91.1 95.4 CD8 Tim3+ 98.9 98.9 97.396.7 97.1 97.7 98.2 95.7 94.9 96.5 EP11001 M1056 M1058 M1023 FreshThawed Fresh Thawed Fresh Thawed Fresh Thawed T cell Re-REP Re-REPRe-REP Re-REP Re-REP Re-REP Re-REP Re-REP CD4 94.3 98.7 74 75.4 86.587.3 94.4 90.6 CD8 96.3 99.3 98 99 97.3 98.6 99 97.7

TABLE 54 FIG. 61: qPCR and Flow-FISH determination of telomere lengthrepeat Tumor ID M1061 M1062 M1063 M1064 M1065 EP11001 M1056 M1058 M1023qPCR 0.111878 0.135842 0.149685 0.179244 0.151774 0.137738 0.1349040.124137 0.086569 Flow-FISH 9.330236 1215041 8.782231 7.174627 8.9615536112918 9.010615 7.944534 5.766692

Example 20: Novel Cryopreserved Tumor Infiltrating Lymphocytes (Ln-144)Administered to Patients with Metastatic Melanoma

Novel cryopreserved tumor infiltrating lymphocytes (LN-144) administeredto patients with metastatic melanoma demonstrates efficacy andtolerability in a multicenter Phase 2 clinical trial

Introduction:

The safety and efficacy of adoptive cell therapy (ACT) withnon-cryopreserved tumor infiltrating lymphocytes (TIL) has been studiedin hundreds of patients with metastatic melanoma. This multicenterclinical trial was initiated with centrally manufactured TILs (LN-144)as non-cryopreserved and cryopreserved infusion products. Our novelmanufacturing process for the non-cryopreserved LN-144 is used in Cohort1, and a shortened 3 weeks, cryopreserved LN-144 is used in Cohort 2.The Cohort 2 manufacturing offers a significantly shorter process,coupled with a cryopreserved TIL product which allows for flexibility ofpatient scheduling and dosing. The shorter manufacturing process reducesthe wait time for the patient to receive their TIL product andcryopreservation adds convenience to logistics and delivery to theclinical sites.

Methods:

C-144-01 is a prospective, multicenter study evaluating metastaticmelanoma patients who receive LN-144. Following a non-myeloablativelymphodepletion with Cy/Flu preconditioning regimen, patients receive asingle infusion of LN-144 followed by the administration of IL-2(600,000 IU/kg) up to 6 doses. Patients are evaluated for objectiveresponse as a primary endpoint for up to 24 months.

Results:

We characterize the cryopreserved LN-144 administered to a second cohortof patients, Cohort 2 (N=10) following the same pre- and post-TILinfusion treatment regimen as used for Cohort 1.

Cohort 2 patients were heavily pretreated with increased number of priorlines with all patients having anti-CTLA-4 and anti-PD-1 therapies, andlarger tumor burden (mean SOD: 15.3, 10.9 cm for Cohorts 2, 1). Mediannumber of prior systemic therapies is 4, 3 for Cohorts 2, 1,respectively. An initial analysis of safety data demonstrates comparabletolerability of cryopreserved LN-144. The safety profile for Cohort 1patients receiving the non-cryopreserved LN-144 continues to beacceptable for this late stage patient population. The most common TEAEsobserved in both cohorts by frequency are nausea, anaemia, febrileneutropenia, neutrophil count decreased, platelet count decreased. Earlyreview of efficacy data indicates anti-tumor activity, including PR, tothe TIL therapy observed in patients treated in Cohort 2.

Conclusions:

This represents the first clinical trial in a multicenter setting withcentrally manufactured TIL assessing a novel process for cryopreservedautologous product with a significantly shorter process (approximately 3weeks). Preliminary results indicate the cryopreserved LN-144 as a safeand tolerable therapeutic option for patients with metastatic melanomawho've failed multiple prior therapies, including checkpoint inhibitors.The cryopreserved LN-144 provides greater flexibility for patients andcaregivers and allows for more immediate treatment for patients withsuch high unmet medical need. NCT02360579.

Example 21: Evaluation of Serum-Free Media for Use in the 2A Process

This example provides data showing the evaluation of the efficacy ofserum-free media as a replacement for the standard CM1, CM2, and CM4media that is currently used in the 2A process. This study testedefficacy of available serum-free media (SFM) and serum free alternativesas a replacement in three phases;

Phase-1: Compared the efficacy of TIL expansion (n=3) using standard vsCTS Optimizer or Prime T CDM or Xvivo-20 serum free media with orwithout serum replacement or platelet lysate.

Phase-2: Tested the candidate serum free media condition in mini-scale2A process using G-Rex 5M (n=3).

Background Information

Though the current media combination used in Pre and Post REP culturehas proven to be effective, REP failures may be occurred with the AIM-V.If an effective serum-free alternative were identified, it would be makethe process more straight-forward and simple to be performed in CMOs byreducing the number of media types used from 3 to 1. Additionally, SFMreduces the chance of adventitious disease by eliminating the use ofhuman serum. This example provides data that showed supports the use ofserum free media in the 2A processes.

Abbreviations

-   -   μl microliter    -   CM1,2,4 Complete Media 1,2,4    -   CTS OpTimizer SFM Cell Therapy System OpTimizer Serum Free Media    -   g Grams    -   Hr Hour    -   IFU Instructions for Use    -   IL-2 Interleukin-2 Cytokine    -   Min Minute    -   mL Milliliter    -   ° C. degrees Celsius    -   PreREP Pre-Rapid Expansion Protocol    -   REP Rapid Expansion Protocol    -   RT Room Temperature    -   SR Serum Replacement    -   TIL Tumor Infiltrating Lymphocytes

Experiment Design

The Pre-REPs and REPs were initiated as mentioned in LAB-008. Theoverview of this 3 phases of experiment is shown in FIG. 128.

As provide in the chart above, the project was intimated to test theserum free media and supplements in two steps.

Step 1. Selection of serum-free media purveyor. preREP and postREP wereset up to mimic 2A process in G-Rex 24 well plate. PreREP were initiatedby culturing each fragment/well of G-Rex 24 well plate in triplicates orquatraplicates per conditions. REP were initiated on Day 11 by culturing4×10e5 TIL/well of G-Rex 24 well, split on Day 16, harvest on Day 22.CTS OpTimizer, X-Vivo 20, and Prime T-CDM were used as potentialserum-free media alternatives for use in the PreREP and REP. CTS ImmuneSR Serum replacement (Life Technologies) or Platelet lysate serum (SDBB)were added at 3% to SFM. Each conditions were planned to test with atleast 3 tumors in both preREP and postREP to mimic 2A process.

Step 2. Identified candidates were further tested on mini-scale 2Aprocesses per protocol (TP-17-007). Briefly, preREP were initiated byculturing 2 fragments/G-Rex 5M flask in triplicates per condition. REPwere initiated on Day 11 using 2×10e6/G-Rex 5M flask, split on Day 16,harvest on Day 22.

Note: Some tumors were processed and setup to measure multipleparameters in one experiment

Observations

Observed equivalent or statistically better results in cell growth whencomparing a serum-free media to the standard used in the 2A process

Observed similar phenotype, IFN-γ production, and metabolite analysisfrom the TIL grown in serum-free media when compared to the TIL grown inthe standard media used in the 2A process.

Results

Testing the Efficacy of Serum Free Media on Pre and Post REP TILExpansion.

CTS Optimizer+SR (Serum Replacement) showed enhanced preREP TILexpansion and comparable REP TIL expansion. CTS OpTimizer, X-Vivo 20,and Prime T-CDM were added with or without 3% CTS Immune CTS SR, weretested against standard condition. In M1079 and L4026, CTS OpTimizer+CSRcondition showed significantly enhanced preREP TIL expansion (p<0.05)when compared with standard conditions (CM1, CM2, CM4) (FIG. 62A).Conversely, CTS Optimizer without CSR did not help preREP TIL expansion(Appendix-1,2,3). CTS Optimizer+CSR showed comparable TIL expansion inPostREP in the two tumour of 3 tested (Figure-2B). A large amount ofvariation occurred in pre and post REP with the X-Vivo 20 and PrimeT-CDM conditions, while CTS Optimizer was relatively consistent betweenquatraplicates. In addition, SFM added platelet lysate did not enhancepreREP and postREP TIL expansion when compared to standards (FIG. 62A).This findings suggesting that serum replacement is certainly needed toprovide a comparable growth to our standard, CTS optimizer+CSR may be acandidate.

Testing candidate condition in the G-Rex 5M mini scale (see FIG. 64).

Phenotypic analysis of Post REP TIL. See FIG. 66 and Table 56 below.

TABLE 56 CD8 skewing with CTS OpTimizer Average % CD8+ Standard CTSM1078 11 34 M1079 29.3 43.85 M1080 33.67 54.37 L4020 0.02 0.17 EP1102028.67 25.07 L4030 0.13 0.09 L4026 9.45 34.06 M1092 5.75 52.47 T6030 6652.6

Interferon-Gamma Comparability

Interferon-gamma ELISA (Quantikine). Production of IFN-γ was measuredusing Quantikine ELISA kit by R&D systems. CTS+SR produced comparableamounts of IFN-γ when compared to our standard condition. See, FIG. 67.

Example 22: T-Cell Growth Factor Cocktail IL-2/IL-15/IL-21 EnhancesExpansion and Effector Function of Tumor-Infiltrating T Cells

Adoptive T cell therapy with autologous tumor infiltrating lymphocytes(TIL) has demonstrated clinical efficacy in patients with metastaticmelanoma and cervical carcinoma. In some studies, better clinicaloutcomes have positively correlated with the total number of cellsinfused and/or percentage of CD8+ T cells. Most current productionregimens solely utilize IL-2 to promote TIL growth. Enhanced lymphocyteexpansion has been reported using IL-15 and IL-21-containing regimens.This study describes the positive effects of adding IL-15 and IL-21 tothe second generation IL-2-TIL protocol recently implemented in theclinic.

Materials and Methods

The process of generating TIL includes a pre-Rapid Expansion Protocol(pre-REP), in which tumor fragments of 1-3 mm³ size are placed in mediacontaining IL-2. During the pre-REP, TIL emigrate out of the tumorfragments and expand in response to IL-2 stimulation.

To further stimulate TIL growth, TIL are expanded through a secondaryculture period termed the Rapid Expansion Protocol (REP) that includesirradiated PBMC feeders, IL-2 and anti-CD3. In this study, a shortenedpre-REP and REP expansion protocol was developed to expand TIL whilemaintaining the phenotypic and functional attributes of the final TILproduct.

This shortened TIL production protocol was used to assess the impact ofIL-2 alone versus a combination of IL2/IL-15/IL-21. These two cultureregimens were compared for the production of TIL grown from colorectal,melanoma, cervical, triple negative breast, lung and renal tumors. Atthe completion of the pre-REP, cultured TIL were assessed for expansion,phenotype, function (CD107a+ and IFNγ) and TCR Vβ repertoire.

pre-REP cultures were initiated using the standard IL-2 (600 IU/ml)protocol, or with IL-15 (180 IU/ml) and IL-21 (IU/ml) in addition toIL-2. Cells were assessed for expansion at the completion of thepre-REP. A culture was classified as having an increase expansion overthe IL-2 if the overall growth was enhanced by at least 20%. Themelanoma and lung phenotypic and functional studies are presentedherein. See, Table 57 below.

TABLE 57 Enhancement in expansion during the pre-REP withIL-2/IL-15/IL-21 in multiple indications # of IL-2 versus # of studiesdemonstrating >20% IL-2/IL-15/IL-21 enhancement of growth using IL-Tumor Histology studies 2/IL-15/IL-21 (compared to IL-2) Melanoma 5 1/5(20%)  Lung 8 3/8 (38%)  Colorectal 11 7/11 (63%)  Cervical 1 1/1 (100%)Pancreatic 2 2/2 (100%) Glioblastoma 1 1/1 (100%) Triple Negative 1 1/2(50%)  Breast

These data demonstrate an increased TIL product yield when TIL werecultured with IL-2/IL15/IL-21 as compared to IL-2 alone, in addition tophenotypic and functional differences in lung.

The effect of the triple cocktail on TIL expansion wasindication-specific and benefited most the low yield tumors.

The CD8+/CD4+ T cell ratio was increased by the treatment in NSCLC TILproduct.

T cell activity appeared enhanced by the addition of IL-15 and IL-21 toIL-2, as assessed by CD107a expression levels in both melanoma andNSCLC.

The data provided here shows that TIL expansion using a shorter, morerobust process, such as the 2A process described herein in theapplication and other examples, can be adapted to encompassing theIL-2/IL-15/IL-21 cytokine cocktail, thereby providing a means to furtherpromote TIL expansion in particularly in specific indications.

Ongoing experiments are further evaluating the effects ofIL-2/IL-15/IL-21 on TIL function.

Additional experiments will evaluate the effect of the triple cocktailduring the REP (first expansion).

These observations are especially relevant to the optimization andstandardization of TIL culture regimens necessary for large-scaremanufacture of TIL with the broad applicability and availabilityrequired of a main-stream anti-cancer therapy.

Example 23: A Cryopreserved TIL Generated with an Abbreviated Method

Background

This example provides data related to a cryopreserved tumor infiltratinglymphocyte (TIL) product for LN-144, generated with an abbreviatedmethod suitable for high throughput commercial manufacturing exhibitsfavorable quality attributes for adoptive cell transfer (ACT).

Existing methods for generating clinical TIL products involve openoperator interventions followed by extended incubation periods togenerate a therapeutic product. The Generation 1 process takesapproximately 6 weeks and yields a fresh product. To bring TIL therapyto all patients that may benefit from its potential, an abbreviated 22day culture method, Generation 2, suitable for centralized manufacturingwith a cryopreserved drug product capable of shipment to distantclinical sites was developed. Generation 2 represents a flexible,robust, closed, and semi-automated cell production process that isamenable to high throughput manufacturing on a commercial scale. Drugproducts generated by this method have comparable quality attributes tothose generated by the Generation 1 process.

Study Objectives:

Drug products generated by Generation 1 (a process 1C embodiment) andGeneration 2 (a process 2A embodiment) processes were assayed todetermine comparability in terms of the following quality attributes:

-   -   Dose and fold expansion.    -   T-cell purity and proportions of T-cell subsets.    -   Phenotypic expression of co-stimulatory molecules on T-cell        subsets.    -   Average relative length of telomere repeats.    -   Ability to secrete cytokine in response to TCR reactivation.    -   T-cell receptor diversity.        Overview of TIL Therapy Process:

EXTRACTION: Patient's TIL are removed from suppressive tumormicroenvironment (via surgical resection of a lesion)

EXPANSION: TIL expanded exponentially in culture with IL-2 to yield10⁹-10¹¹ TIL, before infusing them into the patient

PREPARATION: Patient receives NMA-LD (non-myeloablative lymphodepletion,cyclophosphamide: 60 mg/kg, IV×2 doses and fludarabine: 25 mg/m²×5doses) to eliminate potentially suppressive tumor microenvironment andmaximize engraftment and potency of TIL therapy

INFUSION: Patient is infused with their expanded TIL (LN-144) and ashort duration of high-dose of IL-2 (600,000 IU/kg for up to 6 doses) topromote activation, proliferation, and anti-tumor cytolytic activity ofTIL

TABLE 58 Summary of Process Improvements in Generation 2 ManufacturingProcess Step Gen 1 Gen 2 Impact Fragment ≤21 days, multiple ≤11 days,single closed Shortens culture, Culture bioreactors, multiplebioreactor, no intervention reduces operator interventionsinterventions, amenable to automation. TIL selection IL-2 expanded TIL≤200 × 10⁶ Bulk TIL direct Shorten process by cryopreserved, tested, toco-culture allowing increased selection based on seeding of co-phenotype, thaw, ≤30 × 106 culture, reduces TIL to co-culture steps,eliminates testing Rapid ≤36 Bioreactors, 14 days ≤5 Bioreactors, 11days Reduces operator Expansion interventions, closed system, shortensprocess, amenable to automation. Harvest/Wash Manual open volume Closedsemi-automated Reduces operator reduction and harvest. volume reductionand interventions, Manual wash and harvest. Automated wash automated,concentration by and concentration. maintains closed centrifugation.system. Formulation Fresh hypothermic product Cryopreserved product(≤−150° C.) Shipping flexibility, (2-8° C.) patient scheduling, easierrelease testing, global trials Manufacturing 38 day process time 22 dayprocess time Turnaround to Time patient, clean room throughput, COGsAnalytical Methods and Instrumentation:

Dose and Viability: Final formulated products were sampled and assayedfor total nucleated cells, total viable cells, and viability determinedby acridine orange/DAPI counterstain using the NC-200 automated cellcounter.

Flow cytometry: Formulated drug products were sampled and assayed foridentity by FACS. Percent T-cells was determined as the CD45, CD3 doublepositive population of viable cells. Frozen satellite or sentinel vialsfor each process were thawed and assayed for extended phenotypic markersincluding CD3, CD4, CD8, CD27, and CD28.

Average relative length of telomere repeats: Flow-FISH technology wasused to measure average length of telomere repeat. This assay wascompleted as described in the DAKO® Telomere PNA Kit/FITC for FlowCytometry protocol. Briefly, 2×10⁶ TIL cells were combined with 2×10⁶1301 leukemia cells. The DNA was denatured at 82° C. for 10 minutes andthe PNA-FITC probe was hybridized in the dark overnight at roomtemperature. Propidium Iodide was used to identify the cells in G0/1phase.

Immunoassays: The ability of the drug product to secrete cytokine uponreactivation was measured following co-culture with mAb-coated beads(Life Technologies, anti-CD3, anti-CD28 & anti-CD137). After 24 hrsculture supernatants were harvested frozen, thawed, and assayed by ELISAusing Quantikine IFNγ ELISA kit (R&D systems) according tomanufacturer's instructions.

T-cell receptor diversity: RNA from final formulated products wasisolated and subjected to a multiplex PCR with VDJ specific primers.CDR3 sequences expressed within the TIL product were semi-quantitativelyamplified to determine the frequency and prevalence of unique TILclones. Sequencing was performed on the Illumina MiSeq benchtopsequencer. Values were indexed to yield a score representative of therelative diversity of T-cell receptors in the product.

Results and Conclusions:

Results are provided in FIGS. 75 through 81.

The Generation 2 process produces a TIL product with comparable qualityattributes to Generation 1.

Generation 2 produces similar quantities of highly pure TIL productsthat are composed similar proportions of T-cell subsets expressingcomparable levels of co-stimulatory molecules relative to Gen 1.

Generation 2 TIL display increased diversity of TCR receptors which,when engaged, initiate robust secretion of cytokine.

The cryopreserved drug product introduces critical logisticalefficiencies allowing flexibility in distribution.

Unlike prior processes, the Generation 2 abbreviated 22-day expansionplatform presents a scalable and logistically feasible TIL manufacturingplatform that allows for the rapid generation of clinical scale dosesfor patients in urgent need of therapy.

The Generation 2 TIL manufacturing protocol addresses many of thebarriers that have thus far hindered the wider application of TILtherapy.

Example 24: Evaluating a Range of Allogeneic Feeder Cell:TIL Ratios from100:1 to 25:1

This study tested the proliferation of TIL at 25:1 and 50:1 against thecontrol of 100:1 allogeneic feeder cells to TIL currently utilized inProcess 1C.

Studies published by the Surgery Branch at the National Cancer Institutehave shown the threshold for optimal activation of TIL in the G-Rex 100flask at 5×10⁶ allogeneic feeder cells per cm² at the initiation of theREP⁽¹⁾. This has been verified through mathematical modeling, and, withthe same model, predicted that with a feeder layer optimized forcell:cell contact per unit area the proportion of allogeneic feedercells relative to TIL may be decreased to 25:1 with minimal effect onTIL activation and expansion.

This study established an optimal density of feeder cells per unit areaat REP onset, and validated the effective range of allogeneic feederratios at REP initiation needed to decrease and normalize the amount offeeder cells used per clinical lot. The study also validated theinitiation of the REP with less than 200×10⁶ TIL co-cultured with afixed number of feeder cells.

A. Volume of a T-cell (10 μm diameter): V=(4/3) πr³=523.6 μm³

B. Column of G-Rex 100 (M) with a 40 μm (4 cells) height: V=(4/3)πr³=4×10¹² μm³

C. Number cell required to fill column B: 4×10¹² μm³/523.6 μm³=7.6×10⁸μm³*0.64=4.86×10⁸

D. Number cells that can be optimally activated in 4D space:4.86×10⁸/24=20.25×10⁶

E. Number of feeders and TIL extrapolated to G-Rex 500: TIL: 100×10⁶ andFeeder: 2.5×10⁹

Equation 1. Approximation of the number of mononuclear cells required toprovide an icosahedral geometry for activation of TIL in a cylinder witha 100 cm² base. The calculation derives the experimental result of˜5×10⁸ for threshold activation of T-cells which closely mirrors NCIexperimental data.⁽¹⁾ (C) The multiplier (0.64) is the random packingdensity for equivalent spheres as calculated by Jaeger and Nagel in1992⁽²⁾. (D) The divisor 24 is the number of equivalent spheres thatcould contact a similar object in 4 dimensional space “the Newtonnumber.”⁽³⁾.

REFERENCES

-   ⁽¹⁾Jin, Jianjian, et. al., Simplified Method of the Growth of Human    Tumor Infiltrating Lymphocytes (TIL) in Gas-Permeable Flasks to    Numbers Needed for Patient Treatment. J Immunother. 2012 April;    35(3): 283-292.-   ⁽²⁾Jaeger H M, Nagel S R. Physics of the granular state. Science.    1992 Mar. 20; 255(5051):1523-31.-   ⁽³⁾ O. R. Musin (2003). “The problem of the twenty-five spheres”.    Russ. Math. Surv. 58 (4): 794-795.

Example 25: Studies of Key Quality Attributes for TIL Product

Background

Adoptive T-cell therapy with autologous tumor infiltrating lymphocytes(TIL) has demonstrated clinical efficacy in patients with metastaticmelanoma and other tumors¹⁻³.

Most reports from clinical studies have included exploratory analyses ofthe infused TIL products with the intention of identifying qualityattributes such as sterility, identity, purity, and potency that couldrelate to product efficacy and/or safety.^(4,5)

Here we present the evaluation of three key product parameters from theTIL product that may contribute to a future quality control platform foruse in the commercial manufacture of TIL.

Overview of TIL Therapy Process

1. The tumor was excised from the patient and transported to the GMPManufacturing facility.

2. Upon arrival the tumor is fragmented and placed in flasks with IL-2for a pre-Rapid Expansion Protocol (REP).

3. pre-REP TIL were further propagated in a REP protocol in the presenceof irradiated PBMCs, anti-CD3 antibody (30 ng/mL), and IL-2 (3000IU/mL).

4. TIL products were assessed for critical quality attributes including:(1) Identity (2) Purity, and (3) Potency.

5. Prior to infusion of expanded TIL (LN-144), patient received anon-myeloablative lymphodepletion regimen consisting of cyclophosphamideand fludarabine. Following infusion of TIL, patients received a shortduration (up to 6 doses) of high-dose IL-2 (600,000 IU/kg) to supportgrowth and engraftment of transferred TIL.

Study Objectives

Goal: To fully characterize TIL products for identity, purity, andpotency, and thereby (a) guide the definition of critical qualityattributes and (b) support the establishment of formal release criteriato be implemented in commercial production of TIL products.

Strategy: To develop the following analytical methodologies to supportTIL product characterization. In particular, the following methods wereperformed: phenotypic analysis by flow cytometry for an identity andpurity assessment, residual tumor cell detection assay for a measure ofpurity, and interferon-gamma release assay for assessment of potency.

Materials & Methods

Identity and Purity

Phenotypic characterization: TIL products were stained with anti-CD45,anti-CD3, anti-CD8, anti-CD4, anti-CD45RA, anti CCR7, anti CD62L,anti-CD19, anti-CD16, and anti-CD56 antibodies and analyzed by flowcytometry for the quantification of T and non-T cell subsets.

Purity

Residual tumor detection assay: TIL products were stained with anti-MCSP(melanoma-associated chondroitin sulfate proteoglycan) and anti-CD45antibodies, as well as a Live/Dead fixable Aqua dye, then analyzed byflow cytometry for the detection of melanoma cells. Spiked controls wereused to assess accuracy of tumor detection and to establish gatingcriteria for data analysis.

Potency

IFNγ release assay: TIL products were re-stimulated withanti-CD3/CD28/CD137 coated beads for 18 to 24 hours after whichsupernatants were harvested for assessment of IFNγ secretion using anELISA assay.

Results

Identity: The majority (>99%) of melanoma TIL product was composed ofCD45⁺CD3⁺ cells

FIGS. 86A-86C provides phenotypic characterization of TIL products using10-color flow cytometry assay. (A) Percentage of T-cell and non-T-cellsubsets was defined by CD45⁺CD3⁺ and CD45-(non-lymphocyte)/CD45⁺CD3⁻(non-T-cell lymphocyte), respectively. Overall, >99% of the TIL productstested consisted of T-cell (CD45⁺CD3⁺). Shown is an average of TILproducts (n=10). (B) Percentage of two T-cell subsets includingCD45⁺CD3⁺CD8⁺ (blue open circle) and CD45⁺CD3⁺CD4⁺ (pink open circle).No statistical difference in percentage of both subsets was observedusing student's unpaired T test (P=0.68). (C) Non-T-cell population wascharacterized for four different subsets including: 1) Non-lymphocyte(CD45⁻), 2) NK cell (CD45⁺CD3⁻CD16⁺/56⁺), 3) B-cell (CD45⁺CD19⁺), and 4)Non-NK/B-cell (CD45⁺CD3⁻CD16⁻CD56⁻CD19⁻).

Identity: The majority of melanoma TIL product exhibited effector ormemory T-cell phenotype, associated with T-cell cytotoxic function.

FIGS. 87A and 87B show the characterization of T-cell subsets inCD45⁺CD3⁺CD4⁺ and CD45⁺CD3⁺CD8⁺ cell populations. Naïve, central memory(TCM), effector memory (TEF), and effector memory RA⁺ (EMRA) T-cellsubsets were defined using CD45RA and CCR7. FIGS. 87A and 87B showrepresentative T-cell subsets from 10 final TIL products in both CD4⁺(A), and CD8⁺ (B) cell populations. Effector memory T-cell subset (blueopen circle) were a major population (>93%) in both CD4⁺ and CD8⁺subsets of TIL final product. Less than 7% of the TIL products cellswere central memory subset (pink open circle). EMRA (gray open circle)and naïve (black open circle) subsets were barely detected in TILproduct (<0.02%). p values represent the difference between EM and CMusing student's unpaired T test.

Purity: MCSP represents an appropriate melanoma tumor marker for purityassay.

FIGS. 88A and 88B show the detection of MCSP and EpCAM expression inmelanoma tumor cells. Melanoma tumor cell lines (WM35, 526, and 888),patient-derived melanoma cell lines were generated according to themethods described herein (1028, 1032, and 1041), and a colorectaladenoma carcinoma cell line (HT29 as a negative control) werecharacterized by staining for MCSP (melanoma-associated chondroitinsulfate proteoglycan) and EpCAM (epithelial cell adhesion molecule)markers. (A) Average of 90% of melanoma tumor cells expressed MCSP. (B)EpCAM expression was not detected in melanoma tumor cell lines ascompared positive control HT29, an EpCAM+ tumor cell line.

Purity: Development of a flow cytometry-based assay for detection ofresidual tumor cells in TIL products.

FIGS. 89A and 89B illustrate the detection of spiked controls for thedetermination of tumor detection accuracy. The assay was performed byspiking known amounts of tumor cells into PBMC suspensions (n=10).MCSP+526 melanoma tumor cells were diluted at ratios of 1:10, 1:100, and1:1,000, then mixed with PBMC and stained with anti-MCSP and anti-CD45antibodies and live/dead dye and analyzed by flow cytometry. (A)Approximately 3000, 300, and 30 cells were detected in the dilution of1:10, 1:100, and 1:1000, respectively. (B) An average (AV) and standarddeviation (SD) of cells acquired in each condition was used to definethe upper and lower reference limits.

Purity: Qualification of residual tumor detection assay using spikedcontrols

FIGS. 90A and 90B show the repeatability study of upper and lower limitsin spiked controls. Three independent experiments were performed intriplicate to determine the repeatability of spiking assay. (A) Thenumber of MCSP⁺ detected tumor cells were consistently within the rangeof upper and lower reference limits. (B) Linear regression plotdemonstrates the correlation between MCSP⁺ cells and spiking dilutions(R²=0.99) with the black solid line showing the best fit. The green andgray broken lines represent the 95% prediction limits in standard curveand samples (Exp #1 to 3), respectively.

Purity: Melanoma tumor cell contaminants were below the limits of assaydetection in final TIL product.

FIGS. 91A and 91B show the detection of residual melanoma tumor in TILproducts. TIL products were assessed for residual tumor contaminationusing the developed assay (n=15). The median number and percentage ofdetectable MCSP+ events was 2 and 0.0002%, respectively.

Potency: IFNγ secretion by TIL (consistently >1000 pg/ml) demonstratedeffector function of TIL product.

FIG. 92 shows the potency assessment of TIL products following T-cellactivation. IFNγ secretion after re-stimulation with anti-CD3/CD28/CD137in TIL products assessed by ELISA in duplicate (n=5). IFNγ secretion bythe TIL products was significantly greater than unstimulated controlsusing Wilcoxon signed rank test (P=0.02), and consistently >1000 pg/ml.IFNγ secretion >200 pg/ml was considered to be potent. p value<0.05 isconsidered statistically significant.

Conclusion

Key product parameters of identity, purity, and potency of TIL productswere evaluated. TIL products manufactured according to the methodsdescribed herein consisted of greater than 99% CD45+CD3+ T cells. Themajority of CD4+ and CD8+ TIL subsets exhibited an effector-memoryphenotype, associated with T-cell cytotoxic function. A flowcytometry-based assay to detect contaminant melanoma tumor cells infinal TIL product was successfully developed and qualified. Applyingthis assay, contaminant melanoma tumor cells in final TIL product wereshown to be below the limits of assay detection. IFNγ secretion by finalTIL product following anti-CD3/CD28/CD137 re-stimulation may serve as apotency assay for commercially manufactured TIL. These data provide thefoundation of a quality control platform that will support furtherdevelopment of critical quality attributes for commercial production ofTIL products.

Example 26: A Cryopreserved TIL Product Generated with an AbbreviatedMethod Suitable for High Throughput

Commercial Manufacturing Exhibits Favorable Quality Attributes forAdoptive Cell Transfer

Background

Classical methods of generating tumor infiltrating lymphocytes (TIL) foradoptive cell transfer (ACT) involve multiple ex vivo incubation stepsto yield a fresh (non-cryopreserved) infusion product.

The first generation (Gen 1) process produced a dose of fresh TIL inapproximately 6 weeks. A second generation (Gen 2) TIL manufacturingprocess which abbreviates the ex vivo culture duration to 22 days wasdeveloped (FIG. 93).

The Gen 2 process is suitable for centralized manufacturing and yields acryopreserved TIL infusion product that brings convenience inscheduling, logistics, and delivery to the clinical sites. Thecryopreserved TIL infusion product for LN-144 produced by the Gen 2process has comparable quality attributes to the non-cryopreserved TILinfusion product for TILs generated by the Gen 1 method. The Gen 2 TILmanufacturing method represents a flexible, robust, closed, andsemi-automated cell production process that is amenable to highthroughput TIL manufacturing on a commercial scale.

Study Objective

TIL infusion products generated by Gen 1 and Gen 2 manufacturingprocesses were assessed to determine comparability in terms of thefollowing quality attributes: (1) Cell count (dose), viability, growthrate of REP phase, (2) T-cell purity and phenotypic expression ofco-stimulatory molecules on T-cell subsets, (3) Average relative lengthof telomere repeats, (4) Ability to secrete IFNγ in response to CD3,CD28, CD137 engagement, and (5) Diversity of T-cell receptors present inthe final infusion product (FIG. 94).

Analytical Methods & Instrumentation

Cell Count and Viability: Final formulated infusion products weresampled and assayed for total nucleated cells, total viable cells, andviability determined by acridine orange/DAPI counterstain using theNC-200 automated cell counter. Process Development lots were assayed onthe Nexcellom Cellometer K2 Cell Viability Counter using acridineorange/propidium iodine dual florescent staining.

Phenotypic markers: Formulated infusion products were sampled andassayed for identity by immunofluorescent staining. Percent T-cells wasdetermined as the CD45+,CD3+(double positive) population of viablecells. Frozen satellite or sentinel vials for each process were thawedand assayed for extended phenotypic markers including CD3, CD4, CD8,CD27, and CD28. Fresh infusion products were acquired on the BD FACSCanto II, and extended phenotypic markers on thawed infusion productswere acquired on the Bio-Rad ZE5 Cell Analyzer.

Average relative length of telomere repeats: Flow-FISH technology wasused to measure average length of telomere repeat. This assay wascompleted as described in the DAKO® Telomere PNA Kit/FITC for FlowCytometry protocol. Briefly, 2.0×10⁶ TIL cells were combined with2.0×10⁶ human cell line (1301) leukemia T-cells. The DNA was denaturedat 82° C. for 10 minutes and the PNA-FITC probe was hybridized in thedark overnight at room temperature. Propidium Iodide was used toidentify the cells in G0/1 phase.

Immune function: The ability of the infusion product to secrete IFNγupon reactivation was measured following co-culture with antibody coatedbeads (Life Technologies, anti-CD3, anti-CD28 & anti-CD137). After 24hours culture supernatants were harvested, frozen, thawed, and assayedby ELISA using the Quantikine IFNγ ELISA kit (R&D systems) according tomanufacturer's instructions.

T-cell receptor diversity: RNA from infusion products was isolated andsubjected to a multiplex PCR with VDJ specific primers. CDR3 sequencesexpressed within the TIL product were semi-quantitatively amplified anddeep sequenced to determine the frequency and prevalence of unique TILclones. Sequencing was performed on the Illumina MiSeq benchtopsequencer. Values were indexed to yield a score representative of therelative diversity of T-cell receptors in the product.

Results

On Day 22 the volume reduced cell product was pooled and sampled todetermine culture performance prior to wash and formulation. FIGS.95A-95C shows total viable cells, growth rate, and viability. (A)Samples were analyzed on the NC-200 automated cell counter as previouslydescribed. Total viable cell density is determined by the grand mean ofduplicate counts from 4 independent samples. The Gen 2 process yielded aTIL product of similar dose to Gen 1 (Gen 1 mean=4.10×10¹⁰±2.8×10¹⁰, Gen2 mean=4.12×10¹⁰±2.5×10¹⁰). (B) The growth rate was calculated for theREP phase as. (C) Cell viability was assessed from 9 process developmentlots using the Cellometer K2 as previously described. No significantdecrease in cell viability was observed following a single freeze-thawcycle of the formulated product. Average reduction in viability uponthaw and sampling was 2.19%.

FIGS. 96A-96C show that Gen 2 products are highly pure T-cell cultureswhich express costimulatory molecules at levels comparable to Gen 1.(FIG. 96A) Fresh formulated drug products were assayed for identity byflow cytometry for release. Gen 1 and Gen 2 processes produce highpurity T-cell cultures as defined by CD45+,CD3+(double positive)phenotype. (FIGS. 96B and 96C) Cryopreserved satellite vials offormulated drug product were thawed and assayed for extended phenotypeby flow cytometry as previously described. Gen 1 and Gen 2 productsexpressed similar levels of costimulatory molecules CD27 and CD28 onT-cell subsets. Costimulatory molecules such as CD27 and CD28 may berequired to supply secondary and tertiary signaling necessary foreffector cell proliferation upon T-cell receptor engagement. P-value wascalculated using Mann-Whitney ‘t’ test.

FIG. 97 shows that Gen 2 products trend toward longer relative telomere.Lengths. Flow-FISH technology was used to measure the average length ofthe telomere repeat as previously described. The RTL value indicatedthat the average telomere fluorescence per chromosome/genome in Gen 1was 7.5%±2.1%, and Gen 2 was 8.4%±1.8% of the telomere fluorescence perchromosome/genome in the control cells line (1301 Leukemia cell line).Data indicate Gen 2 products on average have comparable telomere lengthsto Gen 1 products. Telomere length is a surrogate measure of the lengthof ex vivo cell culture.

FIG. 98 shows that Gen 2 drug products secrete IFNγ in response to CD3,CD28, and CD137 engagement. Cryopreserved drug products were thawed andincubated with antibody-coated beaded as previously described. Data isexpressed as the amount of IFNγ produced by 5×10⁵ viable cells in 24hrs. Gen 2 drug products exhibited an increased ability to produce IFNγupon reactivation relative to Gen 1 drug products. The ability of thedrug product to be reactivated and secrete cytokine is a surrogatemeasure of in-vivo function upon TCR binding to cognate antigen in thecontext of HLA.

FIGS. 99A and 99B shows that Gen 2 drug products have an increaseddiversity of unique T-cell receptors. T-cell receptor diversity wasassessed as follows. RNA from 10×10⁶ TIL from Gen 1 and Gen 2 infusionproducts was assayed to determine the total number and frequency ofunique CDR3 sequences present in each product. (FIG. 99A) Unique CDR3sequences were indexed relative to frequency in each product to yield ascore representative of the overall diversity of T-cell receptors in theproduct. (FIG. 99B) The average total number of unique CDR3 sequencespresent in each infusion product. TIL products from both processes werecomposed of polyclonal populations of T-cells with different antigenspecificities and avidities. The breadth of the total T-cell repertoiremay be indicative of the number of actionable epitopes presented ontumor cells.

Conclusions

The Gen 2 manufacturing process produced a TIL infusion product (LN-144)with comparable quality attributes to Gen 1. Gen 2 produced similardoses of highly pure TIL. T-cell subsets were in similar proportions andexpressed costimulatory molecules at comparable levels of relative toGen 1. Gen 2 TIL trended toward longer relative telomere lengthcommensurate with reduced ex vivo culture period. Gen 2 TIL displayed anincreased diversity of TCR receptors which, when engaged, initiatedrobust secretion of IFN-γ, a measure of cytolytic effector function.Thus, the Gen 2 abbreviated 22-day closed expansion process withcryopreserved infusion product presents a scalable and logisticallyfeasible TIL manufacturing platform that allows for the rapid generationof clinical scale doses for cancer patients in immediate need of a noveltherapy option.

REFERENCES

-   ¹Dudley, M. E. et al. Adoptive cell transfer therapy following    non-myeloablative but lymphodepleting chemotherapy for the treatment    of patients with refractory metastatic melanoma. J Clin Oncol 23,    2346-2357, doi:10.1200/JC0.2005.00.240 (2005).-   ²Chandran, S. S. et al. Treatment of metastatic uveal melanoma with    adoptive transfer of tumour-infiltrating lymphocytes: a    single-centre, two-stage, single-arm, phase 2 study. Lancet Oncol,    doi:10.1016/S1470-2045(17)30251-6 (2017).-   ³Stevanovic, S. et al. Complete regression of metastatic cervical    cancer after treatment with human papillomavirus-targeted    tumor-infiltrating T cells. J Clin Oncol 33,    doi:10.1200/jco.2014.58.9093 (2015).-   ⁴FDA Reviewers and Sponsors: Content and Review of Chemistry,    Manufacturing, and Control (CMC) Information for Human Gene Therapy    Investigational New Drug Applications (INDs), 21 CFR 610.3(r), 2008.-   ⁵Richards J O, Treisman J, Garlie N, Hanson J P, Oaks M K. Flow    cytometry assessment of residual melanoma cells in    tumor-infiltrating lymphocyte cultures. Cytometry A 2012; 81:374-81.

Example 27: The T-Cell Growth Factor Cocktail IL-2/IL-15/IL-21 EnhancedExpansion and Effector Function of Tumor-Infiltrating T Cells in a NovelProcess Described Herein

Background

Adoptive T cell therapy with autologous TILs has demonstrated clinicalefficacy in patients with metastatic melanoma and cervical carcinoma. Insome studies, better clinical outcomes have positively correlated withthe total number of cells infused and/or percentage of CD8+ T cells.Most current production regimens solely utilize IL-2 to promote TILgrowth. Enhanced lymphocyte expansion has been reported using IL-15 andIL-21-containing regimens. This study describes the positive effects andsynergies of adding IL-15 and IL-21 to embodiments of process 2A andGeneration 2 TIL manufacturing processes.

Generation of TIL Using a Novel Process Described Herein

The tumor is excised from the patient and transported to the GMPmanufacturing facility or a laboratory for research purposes. Uponarrival the tumor was fragmented, and placed into flasks with IL-2 forpre-Rapid Expansion Protocol (pre-REP) for 11 days. For the triplecocktail studies, IL-2, IL-15, and IL-21 (IL-2/IL-15/IL-21) was added atthe initiation of the pre-REP. For the Rapid Expansion Protocol (REP),TIL were cultured with feeders and anti-CD3 antibody for an additional11 days (FIG. 100).

Materials and Methods

The process of generating TIL included a pre-Rapid Expansion Protocol(pre-REP), in which tumor fragments of 1-3 mm3 size were placed in mediacontaining IL-2. During the pre-REP, TIL emigrated out of the tumorfragments and expand in response to IL-2 stimulation.

To further stimulate TIL growth, TIL were expanded through a secondaryculture period termed the Rapid Expansion Protocol (REP) that includedirradiated PBMC feeders, IL-2 and anti-CD3 antibody. A shortened pre-REPand REP expansion protocol was developed to expand TIL while maintainingthe phenotypic and functional attributes of the final TIL product. Thisshortened TIL-generation protocol was used to assess the impact of IL-2alone versus a combination of IL2/IL-15/IL-21 added to the pre-REP step.These two culture regimens were compared for the generation of TIL grownfrom colorectal, melanoma, cervical, triple negative breast, lung andrenal tumors. At the completion of the pre-REP, cultured TIL wereassessed for expansion, phenotype, function (CD107a+ and IFNγ) and TCRVβ repertoire.

The study shows enhancement in expansion during the pre-REP withIL-2/IL-15/IL-21 in multiple tumor histologies. Pre-REP cultures wereinitiated using the standard IL-2 (6000 IU/mL) protocol, or with IL-15(180 IU/mL) and IL-21 (1 IU/mL) in addition to IL-2 (FIG. 101). Cellswere assessed for expansion at the completion of the pre-REP. A culturewas classified as having increased expansion over the IL-2 if theoverall growth was enhanced by at least 20%. Melanoma and lungphenotypic and functional studies are discussed further in the followingparagraphs (bolded text in FIG. 101).

IL-2/IL-15/IL-21 enhanced the percentage of CD8+ cells in lungcarcinoma, but not in melanoma. In FIGS. 102A and 102B, TIL derived from(A) melanoma (n=4), and (B) lung (n=7) were assessed phenotypically forCD4+ and CD8+ cells using flow cytometry post pre-REP. p valuerepresents the difference between the IL-2 and IL-12/IL-15/IL-21conditions using the student's unpaired t-test.

Expression of CD27 was slightly enhanced in CD8+ cells in culturestreated with IL-2/IL-15/IL-21. In FIGS. 103A and 103B, TIL derived from(A) melanoma (n=4), and (B) lung (n=7) were assessed phenotypically forCD27+ and CD28+ in the CD4+ and CD8+ cells using flow cytometry postpre-REP. Expression of CD27, a cellular marker associated with a youngerphenotype that has correlated with outcomes to adoptive T cell therapy,was slightly enhanced in CD8+ TIL derived from culture withIL-2/IL-15/IL-21 vs IL-2 alone.

T cell subsets were unaltered with the addition of IL-15/IL-21. In FIGS.104A and 104B, TIL were assessed phenotypically for effector/memorysubsets (CD45RA and CCR7) in the CD8+ and CD4+(data not shown) cellsfrom (A) melanoma (n=4), and (B) lung (n=8) via flow cytometry postpre-REP. TEM=effector memory (CD45RA−, CCR7−), TCM=central memory(CD45RA−, CCR7+), TSCM=stem cell memory (CD45RA+, CCR7+), TEMRA=effectorT cells (CD45RA+CCR7−).

The functional capacity of TIL was differentially enhanced withIL-2/IL-15/IL-21. In FIGS. 105A and 105B, TIL derived from (A) melanoma(n=4) and (B) lung (n=5) were assessed for CD107a+ expression inresponse to PMA stimulation for 4 hours in the CD4+ and CD8+ cells, byflow cytometry. (C) pre-REP TIL derived from melanoma and lung werestimulated for 24 hours with soluble anti-CD3 antibody and thesupernatants assessed for IFNγ by ELISA.

The relative frequency of the TCRvβ repertoire was altered in responseto IL-2/IL-15/IL-21 in lung, but not in melanoma. In FIGS. 106A and106B, the TCRvβ repertoire (24 specificities) were assessed in the TILderived from a (A) melanoma and (B) lung tumor using the Beckman Coulterkit for flow cytometry.

Summary

This work demonstrates the ability of the IL-2/IL-15/IL-21 cocktail toenhance TIL numbers compared to IL-2 alone (>20%) in the Generation 2process, in addition to impacting phenotypic and functionalcharacteristics.

The effect of the triple cocktail on TIL expansion was histologydependent. The CD8+/CD4+ T cell ratio was increased with the addition ofIL-2/IL-15/IL-21 in lung tumors. Addition of IL-15 and IL-21 enhancedCD107a expression and IFNγ production in TIL derived from lung tumors.The addition of IL-2/IL-15/IL-21 altered the TCRvβ repertoire in thelung. The Generation 2 TIL expansion process was used to encompass theIL-2/IL-15/IL-21 cytokine cocktail, thereby providing a means to furtherpromote TIL expansion in specific tumor histologies, such as lung andcolorectal tumors. These observations are especially relevant to theoptimization and standardization of TIL culture regimens necessary forlarge-scare manufacture of TIL with the broad applicability andavailability required of a main-stream anti-cancer therapy.

Example 28: Novel Cryopreserved Tumor Infiltrating Lymphocytes (LN-144)Administered to Patients with Metastatic Melanoma Demonstrated Efficacyand Tolerability in a Multicenter Phase 2 Clinical Trial

Background

The safety and efficacy of adoptive cell therapy (ACT) utilizing tumorinfiltrating lymphocytes (TIL) has been studied in hundreds of patientswith metastatic melanoma, and has demonstrated meaningful and durableobjective response rates (ORR).¹ In an ongoing Phase 2 trial, C-144-01utilizing centralized GMP manufacturing of TIL, both non-cryopreservedGeneration 1 (Gen 1) and cryopreserved Generation 2 (Gen 2) TILmanufacturing processes were assessed.

Gen 1 is approximately 5-6 weeks in duration of manufacturing(administered in Cohort 1 of C-144-01 study), while Gen 2 is 22 days induration of manufacturing (process 2A, administered in Cohort 2 ofC-144-01 study). Preliminary data from Cohort 1 patients infused withthe Gen 1 LN-144 manufactured product, was encouraging in treatingpost-PD-1 metastatic melanoma patients as the TIL therapy producedresponses.′ Benefits of Gen 2 included: (A) reduction in the timepatients and physicians wait to infuse TIL to patient; (B)cryopreservation permits flexibility in scheduling, distribution, anddelivery; and (C) reduction of manufacturing costs. Preliminary datafrom Cohort 2 is presented herein. FIG. 107 shows an embodiment of theGen 2 cryopreserved LN-144 manufacturing process (process 2A).

Study Design: C-144-01 Phase 2 Trial in Metastatic Melanoma

Phase 2, Multicenter, 3-Cohort Study to Assess the Efficacy and Safetyof Autologous Tumor Infiltrating Lymphocytes (LN-144) for Treatment ofPatients with Metastatic Melanoma.

Key Inclusion Criteria: (1) Measurable metastatic melanoma and ≥1 lesionresectable for TIL generation; (2) Progression on at least one priorline of systemic therapy; (3) Age ≥18; and (4) ECOG PS 0-1.

Treatment Cohorts: (1) Non-Cryopreserved LN-144 product; (2)Cryopreserved LN-144 product; and (3) Retreatment with LN-144 forpatients without response or who progress after initial response. FIG.108 shows the study design.

Endpoints: (1) Primary: Efficacy defined as ORR and (2) Secondary:Safety and Efficacy.

Methods

Cohort 2 Safety Set: 13 patients who underwent resection for the purposeof TIL generation and received any component of the study treatment.

Cohort 2 Efficacy Set: 9 patients who received the NMA-LDpreconditioning, LN-144 infusion and at least one dose of IL-2, and hadat least one efficacy assessment. 4 patients did not have an efficacyassessment at the time of the data cut.

Biomarker data has been shown for all available data read by the date ofthe data cut.

Results

FIG. 109 provides a table illustrating the Comparison PatientCharacteristics from Cohort 1 (ASCO 2017) vs Cohort 2. Cohort 2 has: 4median prior therapies; all patients have received prior anti-PD-1 andanti-CTLA-4; and had higher tumor burden reflected by greater sum ofdiameters (SOD) for target lesions and higher mean LDH at Baseline. FIG.110 provides a table showing treatment emergent adverse events (≥30%).

For Cohort 2 (cryopreserved LN-144), the infusion product and TILtherapy characteristics were (1) mean number of TIL cells infused:37×10⁹, and (2) median number of IL-2 doses administrations was 4.5.FIG. 111 shows the efficacy of the infusion product and TIL therapy forPatients #1 to #8.

FIG. 112 shows the clinical status of response evaluable patients withstable disease (SD) or a better response. A partial response (PR) forPatient 6 was unconfirmed as the patient did not reached the secondefficacy assessment yet. One patient (Patient 9) passed away prior tothe first assessment (still considered in the efficacy set).

Of the 9 patients in the efficacy set, one patient (Patient 9) was notevaluable (NE) due to melanoma-related death prior to first tumorassessment not represented on FIG. 112. Responses were seen in patientstreated with Gen 2. The disease control rate (DCR) was 78%. Time toresponse was similar to Cohort 1. One patient (Patient 3) withprogressive disease (PD) as best response was not included in the swimlane plot.

FIG. 113 shows the percent change in sum of diameters. Patient 9 had nopost-LN-144 disease assessment due to melanoma-related death prior toDay 42. Day-14: % change of Sum of Diameters from Screening to Baseline(Day-14). Day-14 to Day 126: % change of SOD from Baseline.Day-14=Baseline. Day 0=LN-144 infusion.

Upon TIL treatment, an increase of HMGB1 was observed (FIG. 114). PlasmaHMGB1 levels were measured using HMGB1 ELISA kit (Tecan US, Inc). Datashown represents fold change in HMGB1 levels pre (Day-7) and post (Day 4and Day 14) LN-144 infusion in Cohort 1 and Cohort 2 patients (p valueswere calculated using two-tailed paired t-test based on log-transformeddata). Sample size (bold and italicized) and mean (italicized) valuesare shown in parentheses for each time point. HMGB1 is secreted byactivated immune cells and released by damaged tumor cells. Theincreased HMGB1 levels observed after treatment with LN-144 aretherefore suggestive of an immune-mediated mechanism of anti-tumoractivity.

Plasma IP-10 levels were measured using Luminex assay. Data shown inFIG. 115 represents fold change in IP-10 levels pre (Day-7) and post(Day 4 and Day 14) LN-144 infusion in Cohort 1 and Cohort 2 patients (pvalues were calculated using two-tailed paired t-test based onlog-transformed data). Sample size (bold and italicized) and mean(italicized) values are shown in parentheses for each time point. Thepost-LN-144 infusion increase in IP-10 is being monitored to understandpossible correlation with TIL persistence.

Updated data from Cohort 2 (n=17 patients) is reported in FIG. 116 toFIG. 121. In comparison to Cohort 1 and an embodiment of the Gen 1process, which showed a DCR of 64% and an overall response rate (ORR) of29% (N=14), Cohort 2 and an embodiment of the Gen 2 process showed a DCRof 80% and an ORR of 40% (N=10).

Conclusions

Preliminary results from the existing data demonstrate comparable safetybetween Gen 1 and Gen 2 LN-144 TIL products. Administration of TILsmanufactured with the Gen 2 process (process 2A, as described herein)leads to surprisingly increased clinical responses seen in advanceddisease metastatic melanoma patients, all had progressed on anti-PD-1and anti-CTLA-4 prior therapies. The DCR for cohort 2 was 78%.

Preliminary biomarker data is supportive of the cytolytic mechanism ofaction proposed for TIL therapy.

The embodiment of the Gen 2 manufacturing process described herein takes22 days. This process significantly shortens the duration of time apatient has to wait to receive their TIL, offers flexibility in thetiming of dosing the patients, and leads to a reduction of cost ofmanufacturing, while providing other advantages over prior approachesthat allow for commercialization and registration with health regulatoryagencies. Preliminary clinical data in metastatic melanoma using anembodiment of the Gen 2 manufacturing process also indicates asurprising improvement in clinical efficacy of the TILs, as measured byDCR, ORR, and other clinical responses, with a similar time to responseand safety profile compared to TILs manufactured using the Gen 1process. The unexpectedly improved efficacy of Gen 2 TIL product is alsodemonstrated by a more than five-fold increase in IFN-γ production (FIG.98), which is correlated with improved efficacy in general (FIG. 122),significantly improved polyclonality (FIG. 99A and FIG. 99B), and higheraverage IP-10 and MCP-1 production (FIG. 123 to FIG. 126). Surprisingly,despite the much shorter process of Gen 2, many other criticalcharacteristics of the TIL product are similar to those observed usingmore traditional manufacturing processes, including relative telomerelength (FIG. 97) and CD27 and CD28 expression (FIG. 96B and FIG. 96C).

REFERENCES

-   ¹Goff, et al. Randomized, Prospective Evaluation Comparing Intensity    of Lymphodepletion Before Adoptive Transfer of Tumor-Infiltrating    Lymphocytes for Patients With Metastatic Melanoma. J Clin Oncol.    2016 Jul. 10; 34(20):2389-97.-   ²Sarnaik A, Kluger H, Chesney J, et al. Efficacy of single    administration of tumor-infiltrating lymphocytes (TIL) in heavily    pretreated patients with metastatic melanoma following checkpoint    therapy. J Clin Oncol. 2017; 35 [suppl; abstr 3045].

Example 29: HNSCC and Cervical Carcinoma Phase 2 Studies

Enrollment for the HNSCC (head and neck squamous cell carcinoma;C-145-03) phase 2 study. 13 patients consented to the study, TILs wereharvested from 10 patients and ultimately 7 patients were infused with 1more in progress.

Enrollment in the cervical carcinoma phase 2 study (C-145-04). 8patients consented to the study, TILs were harvested from 4 patients andultimately 2 patients were infused and 2 more in process.

The initial data from the ongoing study is provided in FIG. 127. Stabledisease (SD) and or progressive response was observed in both HCNSCC andcervical cancer patients treated with the TIL therapy at up to 84 days.

The examples set forth above are provided to give those of ordinaryskill in the art a complete disclosure and description of how to makeand use the embodiments of the compositions, systems and methods of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention. Modifications of the above-described modesfor carrying out the invention that are obvious to persons of skill inthe art are intended to be within the scope of the following claims. Allpatents and publications mentioned in the specification are indicativeof the levels of skill of those skilled in the art to which theinvention pertains.

All headings and section designations are used for clarity and referencepurposes only and are not to be considered limiting in any way. Forexample, those of skill in the art will appreciate the usefulness ofcombining various aspects from different headings and sections asappropriate according to the spirit and scope of the invention describedherein.

All references cited herein are hereby incorporated by reference hereinin their entireties and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

Many modifications and variations of this application can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. The specific embodiments and examplesdescribed herein are offered by way of example only, and the applicationis to be limited only by the terms of the appended claims, along withthe full scope of equivalents to which the claims are entitled.

Sequences

-   SEQ ID NO:1 is the amino acid sequence of the heavy chain of    muromonab.-   SEQ ID NO:2 is the amino acid sequence of the light chain of    muromonab.-   SEQ ID NO:3 is the amino acid sequence of a recombinant human IL-2    protein.-   SEQ ID NO:4 is the amino acid sequence of aldesleukin.-   SEQ ID NO:5 is the amino acid sequence of a recombinant human IL-4    protein.-   SEQ ID NO:6 is the amino acid sequence of a recombinant human IL-7    protein.-   SEQ ID NO:7 is the amino acid sequence of a recombinant human IL-15    protein.-   SEQ ID NO:8 is the amino acid sequence of a recombinant human IL-21    protein.    Amino Acid Sequences of Muromonab.

Identifier Sequence (One-Letter Amino Acid Symbols) SEQ ID NO: 1QVQLQQSGAE LARPGASVKM SCKASGYTFT RYTMHWVKQR PGQGLEWIGY INPSRGYTNY  60Muromonab heavyNQKFKDKATL TTDKSSSTAY MQLSSLTSED SAVYYCARYY DDHYCLDYWG QGTTLTVSSA 120chain KTTAPSVYPL APVCGGTTGS SVTLGCLVKG YFPEPVTLTW NSGSLSSGVH TFPAVLQSDL180 YTLSSSVTVT SSTWPSQSIT CNVAHPASST KVDKKIEPRP KSCDKTHTCP PCPAPELLGG240 PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN300 STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE360 LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW420 QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 450 SEQ ID NO: 2QIVLTQSPAI MSASPGEKVT MTCSASSSVS YMNWYQQKSG TSPKRWIYDT SKLASGVPAH  60Muromonab lightFRGSGSGTSY SLTISGMEAE DAATYYCQQW SSNPFTFGSG TKLEINRADT APTVSIFPPS 120chain SEQLTSGGAS VVCFLNNFYP KDINVKWKID GSERQNGVLN SWTDQDSKDS TYSMSSTLTL180 TKDEYERHNS YTCEATHKTS TSPIVKSFNR NEC 213Amino Acid Sequences of Interleukins.

Identifier Sequence (One-Letter Amino Acid Symbols) SEQ ID NO: 3MAPTSSSTKK TQLQLEHLLL DLQMILNGIN NYKNPKLTRM LTFKFYMPKK ATELKHLQCL  60recombinantEEELKPLEEV LNLAQSKNFH LRPRDLISNI NVIVLELKGS ETTFMCEYAD ETATIVEFLN 120human IL-2 RWITFCQSII STLT 134 (rhIL-2) SEQ ID NO: 4PTSSSTKKTQ LQLEHLLLDL QMILNGINNY KNPKLTRMLT FKFYMPKKAT ELKHLQCLEE  60AldesleukinELKPLEEVLN LAQSKNFHLR PRDLISNINV IVLELKGSET TFMCEYADET ATIVEFLNRW 120ITFSQSIIST LT 132 SEQ ID NO: 5MHKCDITLQE IIKTLNSLTE QKTLCTELTV TDIFAASKNT TEKETFCRAA TVLRQFYSHH  60recombinantEKDTRCLGAT AQQFHRHKQL IRFLKRLDRN LWGLAGLNSC PVKEANQSTL ENFLERLKTI 120human IL-4 MREKYSKCSS 130 (rhIL-4) SEQ ID NO: 6MDCDIEGKDG KQYESVLMVS IDQLLDSMKE IGSNCLNNEF NFFKRHICDA NKEGMFLFRA  60recombinantARKLRQFLKM NSTGDFDLHL LKVSEGTTIL LNCTGQVKGR KPAALGEAQP TKSLEENKSL 120human IL-7 KEQKKLNDLC FLKRLLQEIK TCWNKILMGT KEH 153 (rhIL-7)SEQ ID NO: 7MNWVNVISDL KKIEDLIQSM HIDATLYTES DVHPSCKVTA MKCFLLELQV ISLESGDASI  60recombinant HDTVENLIIL ANNSLSSNGN VTESGCKECE ELEEKNIKEF LQSFVHIVQM FINTS115 human IL-15 (rhIL-15) SEQ ID NO: 8MQDRHMIRMR QLIDIVDQLK NYVNDLVPEF LPAPEDVETN CEWSAFSCFQ KAQLKSANTG  60recombinantNNERIINVSI KKLKRKPPST NAGRRQKHRL TCPSCDSYEK KPPKEFLERF KSLLQKMIHQ 120human IL-21 HLSSRTHGSE DS 132 (rhIL-21)

What is claimed is:
 1. A method for expanding tumor infiltratinglymphocytes (TILs) into a therapeutic population of TILs, the methodcomprising: (a) obtaining a first population of TILs from a tumorresected from a subject by processing a tumor sample obtained from thesubject into a tumor digest; (b) adding the tumor digest into a closedsystem; (c) performing a first expansion by culturing the firstpopulation of TILs in a cell culture medium comprising IL-2 to produce asecond population of TILs, wherein the first expansion is performed in aclosed container providing a first gas-permeable surface area, whereinthe first expansion is performed for about 3-11 days to obtain thesecond population of TILs, and wherein the transition from step (b) tostep (c) occurs without opening the system; (d) performing a secondexpansion by supplementing the cell culture medium of the secondpopulation of TILs with additional IL-2, OKT-3, and antigen presentingcells (APCs), to produce a third population of TILs, wherein the secondexpansion is performed for about 7-11 days to obtain the thirdpopulation of TILs, wherein the second expansion is performed in aclosed container providing a second gas-permeable surface area, andwherein the transition from step (c) to step (d) occurs without openingthe system; (e) harvesting the therapeutic population of TILs obtainedfrom step (d), wherein the transition from step (d) to step (e) occurswithout opening the system; (f) transferring the harvested TILpopulation from step (e) to an infusion bag, wherein the transfer fromstep (e) to (f) occurs without opening the system; and (g)cryopreserving the infusion bag comprising the harvested TIL populationfrom step (f) using a cryopreservation process.
 2. The method accordingto claim 1, wherein processing a tumor sample obtained from the subjectinto a tumor digest in step (a) comprises incubating the tumor sample inan enzymatic media.
 3. The method according to claim 2, whereinprocessing a tumor sample obtained from the subject into a tumor digestin step (a) further comprises disrupting the tumor sample mechanicallyso as to dissociate the tumor sample.
 4. The method according to claim3, wherein processing a tumor sample obtained from the subject into atumor digest in step (a) further comprises purifying the disassociatedtumor sample using a density gradient separation.
 5. The methodaccording to claim 2, wherein the enzymatic media comprises DNase. 6.The method according to claim 5, wherein the enzymatic media comprises30 units/mL of DNase.
 7. The method according to claim 2, wherein theenzymatic media comprises collagenase.
 8. The method according to claim7, wherein the enzymatic media comprises 1.0 mg/mL of collagenase. 9.The method according to claim 1, wherein the cell culture medium is CTSOptimizer.
 10. The method according to claim 1, wherein the medium inthe first expansion and/or the second expansion is free of human serum.11. The method according to claim 1, wherein the therapeutic populationof TILs harvested in step (e) comprises sufficient TILs for use inadministering a therapeutically effective dosage to a subject.
 12. Themethod according to claim 11, wherein the number of TILs sufficient foradministering a therapeutically effective dosage is from about 1×10⁹ toabout 9×10¹⁰.
 13. The method according to claim 1, wherein the APCs areperipheral blood mononuclear cells (PBMCs).
 14. The method according toclaim 1, wherein the therapeutic population of TILs harvested in step(e) exhibits an increased subpopulation of CD8+ cells relative to thefirst and/or second population of TILs.
 15. The method according toclaim 13, wherein the PBMCs are supplemented at a ratio of about 1:25TIL:PBMCs.
 16. The method according to claim 1, wherein the firstexpansion in step (c) and the second expansion in step (d) are eachindividually performed within a period of 11 days.
 17. The methodaccording to claim 1, wherein steps (a) through (f) are performed inabout 10 days to about 22 days.
 18. The method according to claim 1,wherein steps (a) through (f) are performed in about 15 days to about 22days.
 19. The method according to claim 1, wherein steps (a) through (f)are performed in about 20 days to about 22 days.
 20. The methodaccording to claim 1, wherein the second population of TILs is at least50-fold greater in number than the first population of TILs.